Hyaluronidase variants with improved stability and pharmaceutical composition comprising the same

Disclosed are novel PH20 variants or fragments thereof with improved thermal stability and enzymatic activity of human hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, and more particularly novel PH20 variants or fragments thereof including one or more amino acid residue substitutions in the variant having the amino acid sequence of SEQ ID NO: 3, wherein one or more amino acid residues at the N-terminus and/or the C-terminus are optionally further deleted.

1. 11. A PH20 variant, wherein the amino acid sequence of the PH20 variant is SEQ ID NO: 3 with modifications consisting of: (a) an amino acid residue substitution of: Y357W, M358V, M358R, L362A, W342I, or E343V relative to SEQ ID NO: 3; (b) an N-terminus deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39, or M1 to A40 of SEQ ID NO: 3; and (c) a C-terminus deletion, wherein the C-terminus of the PH20 variant ends with an amino acid residue selected from any one of amino acid residues I465 to S490 of SEQ ID NO: 3, and wherein the PH20 variant exhibits higher enzymatic activity compared to mature, wild-type human PH20 under the same conditions.
2. 22. The PH20 variant of claim 1, wherein the amino acid residue substitution is Y357W, M358V, or M358R relative to SEQ ID NO: 3.
3. 33. The PH20 variant of claim 1, wherein the amino acid residue substitution is L362A.
4. 44. The PH20 variant of claim 1, wherein the amino acid residue substitution is W342I or E343V relative to SEQ ID NO: 3.
5. 55. The PH20 variant of claim 1, wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 217, 171, or 207.
6. 66. The PH20 variant of claim 1, wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 172.
7. 77. The PH20 variant of claim 1, wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 198 or 173.
8. 88. A pharmaceutical composition comprising the PH20 variant of claim 1, and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
9. 99. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is formulated for subcutaneous injection.
10. 1010. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises a protein drug and is formulated for subcutaneous injection.
11. 1111. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.
12. 1212. A PH20 variant comprising the amino acid sequence of SEQ ID NO: 217, 171, or 207.
13. 1313. A PH20 variant comprising the amino acid sequence of SEQ ID NO: 172.
14. 1414. A PH20 variant comprising the amino acid sequence of SEQ ID NO: 198 or 173.
15. 1515. A pharmaceutical composition comprising the PH20 variant of claim 12, and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
16. 1616. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is formulated for subcutaneous injection.
17. 1717. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition further comprises a protein drug for treating a disease and is formulated for subcutaneous injection.
18. 1818. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.
19. 1919. A pharmaceutical composition comprising the PH20 variant of claim 13, and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
20. 2020. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is formulated for subcutaneous injection.
21. 2121. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises a protein drug for treating a disease and is formulated for subcutaneous injection.
22. 2222. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.
23. 2323. A pharmaceutical composition comprising the PH20 variant of claim 14, and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
24. 2424. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is formulated for subcutaneous injection.
25. 2525. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition further comprises a protein drug for treating a disease and is formulated for subcutaneous injection.
26. 2626. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.
27. 2727. The PH20 variant of claim 1, wherein: (b) the N-terminus deletion is a deletion of amino acid residues M1 to N37 of SEQ ID NO: 3, or (c) the C-terminus of the PH20 variant ends at F468.
28. 2828. A PH20 variant, wherein the amino acid sequence of the PH20 variant is SEQ ID NO: 3 with modifications consisting essentially of: (a) an amino acid residue substitution of: Y357W, M358V, M358R, L362A, W342I, or E343V relative to SEQ ID NO: 3; (b) an N-terminus deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39, or M1 to A40 of SEQ ID NO: 3; and (c) a C-terminus deletion, wherein the C-terminus of the PH20 variant ends with an amino acid residue selected from any one of amino acid residues I465 to S490 of SEQ ID NO: 3, and wherein the PH20 variant exhibits higher enzymatic activity compared to mature, wild-type human PH20 under the same conditions.
29. 2929. A pharmaceutical composition comprising the PH20 variant of claim 28, and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
30. 3030. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition is formulated for subcutaneous injection.
31. 3131. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition further comprises a protein drug for treating a disease and is formulated for subcutaneous injection.
32. 3232. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.

CROSS-REFERENCE TO RELATED APPLICATIONS
This is a U.S. national phase under 35 USC § 371 of International Patent Application No. PCT/KR2021/000943 filed Jan. 25, 2021, which in turn claims priority under 35 USC § 119 of Korean Patent Application No. 10-2020-0009046 filed Jan. 23, 2020. The disclosures of all such applications are hereby incorporated herein by reference in their respective entireties, for all purposes.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “Q303377_SeqListing ST25.txt” created on Aug. 11, 2025 and is 630,784 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to novel human PH20 variants or fragments thereof having increased enzymatic activity and thermal stability compared to human hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, and more particularly to PH20 variants or fragments thereof, which include one or more amino acid residue substitutions, deletions and/or insertions in hyaluronidase variants having the amino acid sequence of SEQ ID NO: 3, and optionally in which one or more amino acid residues are deleted from the N-terminus and/or C-terminus, a method for producing the same, and a pharmaceutical composition containing the same.
Description of the Related Art
The human skin is composed of the epidermis, the dermis, and a subcutaneous fat layer, and there are six types of glycosaminoglycans in the skin. These glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate.
These glycosaminoglycans are composed of repeating disaccharide sugar units. The number of repeating disaccharide sugar units is different among glycosaminoglycans, but ranges from several hundreds to several thousands. Among the glycosaminoglycans, hyaluronic acid is present in the skin more than half of the amount in the body. Hyaluronic acid is synthesized by hyaluronan synthase present in the cell membrane, is present alone without binding to proteoglycans, and is the only glycosaminoglycan having no sulfate group. Other glycosaminoglycans bind to proteoglycans and have a sulfate group. Hyaluronic acid consists of glucuronic acid and N-acetylglucosamine, alternately linked via β-1,4 and β-1,3 bonds, and is composed of about 5,000 repeating units of these disaccharides. It is known that about one-third (5 g) of hyaluronic acid in the human body is degraded every day.
Hyaluronidases are enzymes that degrade hyaluronic acid present in the extracellular matrix. Six hyaluronidase genes are known in humans: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20/SPAM1. Human Hyal1 and Hyal2 are expressed in most tissues. PH20/SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and the acrosomal membrane. However, HyalPS1 is not expressed, because it is a pseudogene. Hyaluronidases are divided, depending on the method by which hyaluronic acid is cleaved, into three types: enzymes (EC 3.2.1.35) that cleave β-1,4 bonds between N-acetylglucosamine and glucuronic acid by the use of H2O; enzymes (EC 3.2.1.36) that cleave β-1,3 bonds between N-acetylglucosamine and glucuronic acid by the use of H2O; and bacterial hyaluronidases (EC 4.2.99.1) that cleave β-1,4 bonds without using H2O.
The catalytic amino acids of Hyal1 are D129 and E131, which hydrolyze hyaluronic acid by substrate-assisted catalysis. Hyal1 exhibits optimum activity at an acidic pH of 3 to 4, and has no enzymatic activity at a pH of 4.5 or higher. In contrast to Hyal1, PH20 exhibits activity throughout a wide pH range of 3 to 8.
Arming et al. identified that the catalytic amino acids of PH20 are D111 and E113 (Arming et al., 1997). Arming et al. designated Leu as the first amino acid of the PH20, from which a signal peptide or the like is removed, and thus the catalytic amino acids of the PH20 containing the signal peptide correspond to D146 and E148, respectively.
Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing the permeability thereof into tissue (skin). The subcutaneous area of the skin has a neutral pH of about 7.0 to 7.5. Thus, among various types of hyaluronidases, PH20 is widely used in clinical practice (Bookbinder et al., 2006). In examples in which PH20 is used in clinical practice, PH20 is used as an eye relaxant and an anesthetic additive in ophthalmic surgery, and is also co-administered with an antibody therapeutic agent which is injected subcutaneously (Bookbinder et al., 2006). In addition, based on the property of hyaluronic acid, which is overexpressed in tumor cells, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the access of an anticancer therapeutic agent to the tumor cells. In addition, it is also used to promote resorption of body fluids and blood, which are excessively present in tissue.
PH20 was first identified in guinea pig sperm by Lathrop et al., and is also known to be expressed in sperm of different species. Human PH20 gene was cloned by Lin et al. and Gmachl et al. Human PH20 has the amino acid sequence of SEQ ID NO: 1, which consists of 509 amino acid residues, and exhibits 60% amino acid identity with guinea pig PH20 gene. Human PH20 enzyme is encoded from the SPAM1 (sperm adhesion molecule-1) gene, and Ser490 of PH20 is present in the form of being bound to glycosylphosphatidylinositol (GPI) on the surface of the sperm plasma membrane and in the acrosomal membrane. Sperm hydrolyzes hyaluronic acid using PH20 when it penetrates oocytes through the hyaluronan-rich cumulus layer of the oocytes. PH20 is present in an amount corresponding to 1% or less of the amount of proteins in sperm, and has six N-glycosylation sites (N82, N166, N235, N254, N368, and N393).
Currently commercially available PH20 is obtained by extraction from the testes of cattle or sheep. Examples thereof include AMPHADASE® (bovine hyaluronidase) and VITRASE® (sheep hyaluronidase).
Bovine testicular hyaluronidase (BTH) is obtained by removing a signal peptide and 56 amino acids on the C-terminal from bovine wild-type PH20 during post-translational modification. BTH is also a glycoprotein, and has a mannose content of 5% and a glucosamine content of 2.2% based on the total components including amino acids. When animal-derived hyaluronidase is repeatedly administered to the human body at a high dose, a neutralizing antibody can be produced. Since animal-derived hyaluronidase contains other biomaterials in addition to PH20, it may cause an allergic reaction when administered to the human body (Bookbinder et al., 2006). In particular, the production and the use of PH20 extracted from cattle can be limited due to concerns of mad cow disease. In order to overcome this problem, studies on the recombinant protein of human PH20 have been conducted.
Recombinant protein of human PH20 has been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells. The recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in terms of the pattern of N-glycosylation during post-translational modification.
Hyaluronidases, protein structures of which have been identified are Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV). Hyal1 is composed of two domains, a catalytic domain and an EGF-like domain. The catalytic domain is in the form of (β/α)8 in which an alpha-helix and a beta-strand, which characterize the secondary structure of the protein, are each repeated eight times (Chao et al., 2007). The EGF-like domain is completely conserved in variants in which the C-terminus of Hyal1 is spliced differently. The amino acid sequences of Hyal1 and PH20 are 35.1% identical, and the protein structure of PH20 has not yet been found.
A recombinant protein of human PH20 was developed by HALOZYME THERAPEUTIC, INC. and has been sold under the trade name HYLENEX® (Bookbinder et al., 2006; Frost, 2007).
When D146 and E148, which are the catalytic amino acids of PH20, were mutated to asparagine (D146N) and glutamine (E148Q), respectively, there was no enzymatic activity (Arming et al., 1997). In addition, when R246 of PH20 was substituted with glycine, the enzymatic activity was reduced by 90%, and when E319 was substituted with glutamine and R322 was substituted with threonine, the enzymatic activity disappeared. A variant in which 36 amino acids at the C-terminus of PH20 were removed (truncation of amino acids 474-509) exhibited a 75% reduction in enzymatic activity compared to wild-type PH20. This mutant was not secreted extracellularly, but remained in HeLa cells. A mutant in which C-terminal 134 amino acids were removed from PH20 had no enzymatic activity and was not secreted extracellularly. According to Frost et al., the C-terminal 477-483 region of PH20 is essential for soluble expression (Frost, 2007). The activity of full-length PH20 (1-509) or a PH20 variant having a C-terminus truncated at position 467 was merely 10% of a PH20 variant having a C-terminus truncated at one of positions 477 to 483 (Frost, 2007).
Recombinant PH20 is medically used as a carrier to promote subcutaneous delivery of pharmaceuticals, to reduce intraocular pressure in patients with ophthalmic diseases, to delay stenosis after surgery, as a dispersant to improve the activity of chemotherapeutic agents in diseases such as cancer, as an auxiliary therapeutic agent for surgery, and the like.
In particular, in the case of protein drugs, recently, high-dose products with high concentrations ranging from tens of mg to hundreds of mg per 1 mL have been developed, and thus the application of recombinant PH20 as a carrier to promote subcutaneous delivery of such protein drugs is increasing. Such protein drugs may have problems of low physical stability resulting from an increase in viscosity and aggregation of proteins due to the high concentration thereof. In addition, the aggregation of proteins is irreversible, and small amounts of proteins start to aggregate and aggregate form larger clumps (Schon et al., 2015). That is, recombinant PH20 administered in combination undergoes aggregation, thus reducing the stability of protein drugs.
Meanwhile, conventional recombinant PH20 is still insufficient from the aspects of thermal stability and expression level. Therefore, there is great demand in industry for a recombinant hyaluronidase having further improved biological and physico-chemical properties.
REFERENCE


    Arming, S., Strobl, B., Wechselberger, C., and Kreil, G. (1997). In-vitro mutagenesis of PH-20 hyaluronidase from human sperm. Eur. J. Biochem. 247, 810-814.
    Bookbinder, L. H., Hofer, A., Haller, M. F., Zepeda, M. L., Keller, G. A., Lim, J. E., Edgington, T. S., Shepard, H. M., Patton, J. S., and Frost, G. I. (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J. Control. Release 114, 230-241.
    Chao, K. L., Muthukumar, L., and Herzberg, O. (2007). Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 46, 6911-6920.
    Frost, G. I. (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin. Drug Deliv. 4, 427-440. Schön, A., Clarkson, B. R., Siles, R., Ross, P., Brown, R. K., Freire, E. (2015) Denatured state aggregation parameters derived from concentration dependence of protein stability. Anal. Chem. 488, 45-50
    WO 2020/022791A (2020 Jan. 30.)


SUMMARY OF THE INVENTION
Therefore, the present invention has been made in view of the above problems, and it is one object of the present invention to provide a PH20 variant or fragment thereof which is improved in thermal stability, enzyme activity and expression level, compared to wild-type PH20, preferably mature wild-type PH20.
It is another object of the present invention to provide a composition for treating cancer containing the PH20 variant or fragment thereof and a method of treating cancer using the same.
In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a PH20 variant or fragment thereof comprising one or more amino acid residue substitutions, deletions and/or insertions in a hyaluronidase variant having the amino acid sequence of SEQ ID NO: 3, and in which one or more amino acid residues at the N-terminus or C-terminus are selectively deleted.
In accordance with another aspect of the present invention, there are provided a composition for treating cancer containing the PH20 variant or fragment thereof and a method of treating cancer using the same.
Effects of the Invention
The PH20 variants or fragments thereof according to the present invention have increased protein expression levels and show an increase in protein aggregation temperature of 4-11.5° C. or so when expressed in CHO (EXPICHO™) cells so that they are efficiently produced and are imparted with higher thermal stability compared to the mature wild-type PH20.
Further, as the result of a substrate-gel assay, one of tests to measure the activity of hyaluronidase, the PH20 variants or fragments thereof according to the present invention have improved protein refolding so that they are re-natured faster than the mature wild-type PH20, and the original enzymatic activity is maintained regardless of the C-terminal cleavage position.
Furthermore, the PH20 variants or fragments thereof according to the present invention have low immunogenicity, so that they can be repeatedly administered to the human body.



BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the results of SDS-PAGE analysis of various variants based on a PH20 variant having the amino acid sequence of SEQ ID NO: 3. The result of the following SDS-PAGE analysis regarding each variant is obtained by purifying an animal cell culture solution expressing each variant through column chromatography and performing 10% SDS-PAGE analysis on the final purified variant;
More specifically, FIG. 1 in part (A) thereof shows the results of SDS-PAGE regarding variants HM98, HM99, HM130, HM143, HM71, HM100, HM131, HM72, HM101, and HM114;
FIG. 1 in part (B) thereof shows the results of SDS-PAGE regarding variants HM63, HM102, HM115, HM64, HM103, HM116, HM125, HM132, HM65, HM133, HM144, HM104 and HM117;
FIG. 1 in part (C) thereof shows the results of SDS-PAGE gel regarding variants HM66, HM105, HM134, HM76, HM106, HM135, HM136 and HM67;
FIG. 1 in part (D) thereof shows the results of SDS-PAGE regarding variants HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM107, HM118, HM90, HM91, HM92, HM93, HM94 and HM95;
FIG. 1 in part (E) thereof shows the results of SDS-PAGE regarding variants HM73, HM111, HM121, HM139, HM74, HM112 and HM140;
FIG. 1 in part (F) thereof shows the results of SDS-PAGE regarding variants HM75, HM141, HM145, HM70, HM77, HM142, HM78, HM79, HM96, HM146, HM147, HM149 and HM150;
FIG. 2 shows the expressions levels of a mature wild-type PH20 and a Hyal2-variant, a Hyal3-variant and a Hyal4-variant in which the region M345 to 1361 of the mature wild-type PH20 was substituted with corresponding sequences of Hyal2, Hyal3 and Hyal4, respectively, wherein Lane CS of SDS-PAGE is a culture medium sample, Lane FT is an unbound impurity in a HisTag column, and Lane E is a HisTag column eluate;
FIG. 3 shows the results of SDS-PAGE analysis of various variants based on a PH20 variant having the amino acid sequence of SEQ ID NO: 3. The result of the following SDS-PAGE analysis regarding each variant is obtained by purifying an animal cell culture solution expressing each variant through column chromatography and performing 10% SDS-PAGE analysis on the final purified variant;
FIG. 3 in part (A) thereof shows the results of SDS-PAGE regarding variants HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168 and HM169;
FIG. 3 in part (B) thereof shows the results of SDS-PAGE regarding variants HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185 and HM186;
FIG. 3 in part (C) thereof shows the results of SDS-PAGE regarding variants HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204 and HM205;
FIG. 3 in part (D) thereof shows the results of SDS-PAGE regarding variants HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219 and HM220;
FIG. 3 in part (E) thereof shows the results of SDS-PAGE regarding variants HM231, HM232, HM233, HM234, HM235, HM243, HM245 and HM246;
FIG. 3 in part (F) thereof shows the results of SDS-PAGE regarding variants HM254, HM261, HM262, HM263, HM266, HM268, HM271, HM275, HM276, HM279, HM280, HM287 and HM288; and
FIG. 4 shows the results of SDS-PAGE confirming the thermostability of wild-type PH20 (L36-Y482) and variant PH20 (F38-F468) having the amino acid sequence of SEQ ID NO: 3, wherein Lanes A, B, C and D show the results of SDS-PAGE analysis regarding initial wild-type PH20 (Lanes A and C) and the PH20 variant of SEQ ID NO: 3 (Lanes B and D) in a reduced form (Lanes A and B) and a non-reduced form (Lanes C and D), and Lanes E, F, G and H show the results of SDS-PAGE analysis regarding initial wild-type PH20 (Lanes E and G) and variant PH20 of SEQ ID NO: 3 (Lanes F and H) in a reduced form (Lanes E and F) and a non-reduced form (Lanes G and H) after being stored for 7 days at 42° C.



DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as appreciated by those skilled in the field to which the present invention pertains. In general, the nomenclature used herein is well-known in the art and is ordinarily used.
In the present invention, the position of the amino acid residue of each variant is referred from the amino acid sequence according to SEQ ID NO: 1, when described based on wild-type PH20, and the position of the amino acid residue of each variant is referred from the amino acid sequence according to SEQ ID NO: 3, when described based on the PH20 variant having SEQ ID NO: 3.
The present inventors found through previous research that a hyaluronidase PH20 variant, which includes one or more amino acid residue substitutions in the region corresponding to an alpha-helix region and/or a linker region thereof, preferably an alpha-helix 8 region (S347 to C381) and/or a linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8, in wild-type PH20 having an amino acid sequence of SEQ ID NO: 1, preferably mature wild-type PH20, and optionally in which one or more of the N-terminal and/or C-terminal amino acid residues are selectively cleaved and deleted, exhibits superior efficacy compared to conventional wild-type PH20 or fragments thereof, and filed a patent application regarding this finding (see WO 2020/022791A).
As used herein, the term “mature wild-type PH20” means a protein consisting of amino acid residues L36 to Y482 or L36 to S490 of SEQ ID NO: 1, which lack M1 to T35, which form a signal peptide, and N483 to L509 or A491 to L509, which are not related to the substantial enzymatic function of PH20, in the amino acid sequence of SEQ ID NO: 1 of wild-type PH20.
Specifically, the present inventors found through previous research that, when amino acid sites corresponding to T341 to 1361, which is a part of an alpha-helix 8 region (S347 to C381) and/or a linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8, in wild-type PH20 having an amino acid sequence of SEQ ID NO: 1 is substituted with amino acid residues corresponding to wild-type Hyal1 having the sequence of SEQ ID NO: 2, the expression efficiency and enzymatic activity are improved, and fragments in which a part of the amino acid sequence at the N-terminus and C-terminus is deleted also exhibit superior expression efficiency and high enzymatic activity.







TABLE 1





Amino acid sequence of wild-type PH20 and


wild-type Hyal1















Amino acid sequence of wild-type PH20 


(SEQ ID NO: 1)


MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFL


WAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYP


YIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEW


RPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFL


VETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCENVEIKRNDDLS


WLWNESTALYPSTYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPV


FAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKS


CLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHL


NPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVK


DTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILF


LIISSVASL


 


Amino acid sequence of wild-type Hyal1 


(SEQ ID NO: 2)


MAAHLLPICALFLTLLDMAQGFRGPLLPNRPFTTVWNANTQWCLERHGVD


VDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNA


SLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQR


SRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLOLGRALRPRGLWG


FYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPA


VLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPL


DELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILN


VTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSL


RGALSLEDQAQMAVEFKCRCYPGWQAPWCERKSMW









As a result of continuous research, the present inventors found that a variant having the sequence of SEQ ID NO: 3, constructed by substituting the amino acid region corresponding to T341 to 1361 of wild-type PH20 having the amino acid sequence of SEQ ID NO: 1 with the corresponding amino acid sequence of wild-type Hyal1 having the sequence of SEQ ID NO: 2, still exhibits excellent expression efficiency and high enzymatic activity, as well as remarkably improved protein aggregation temperature (Tagg), compared to the wild-type PH20, although it includes additional substitutions, deletions and/or insertions of amino acid residues, and further optionally includes deletions of one or more amino acid residues at the N-terminus and/or C-terminus. Based on this finding, the present invention has been completed.
The variant having the sequence of SEQ ID NO: 3 is constructed by substituting 15 amino acid residues, namely, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1.
In this regard, the PH20 variant or fragment thereof according to the present invention includes substitution, deletion and/or insertion of one or more amino acid residues in the PH20 variant having the amino acid sequence of SEQ ID NO: 3, and optionally includes deletion of one or more amino acid residues at the N-terminus and/or C-terminus.
As described above, the variant having the amino acid sequence of SEQ ID NO: 3 is a variant in which amino acid residues of T341 to 1361 of wild-type PH20 are substituted with corresponding amino acid residues of wild-type Hyal1 (see Table 2). The variant having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof including amino acid residue deletion at the N-terminus and C-terminus was identified as a variant having activity and stability superior to those of wild-type PH20 in previous research.







TABLE 2





Amino acid sequence of PH20 variant in which


amino acid residues at positions T341 to I361 


of wild-type PH20 are substituted with cor-


responding amino acid residues of Hyla1 


(SEQ ID NO: 3)

















MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVI



PNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGV



TIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDIT



FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQN



VQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYL



FPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSTY



LNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVF



TDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQ



AIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSD



YLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYST



LSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNA



SPSTLSATMFIVSILFLIISSVASL










Specifically, the PH20 variant or fragment thereof according to the present invention may include one or more mutations, preferably one or more amino acid residue substitutions, deletions and/or insertions in the amino acid sequence of SEQ ID NO: 3, and has a higher protein aggregation temperature (Tagg), which is an index indicating protein stability, than the wild-type PH20. In addition, the PH20 variant according to the present invention does not include the wild-type PH20 of SEQ ID NO: 1.
As used herein, the term “PH20 variant” is intended to include a variant having not only a mutation of one or more amino acid residues, preferably substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 3, but also deletion of one or more amino acid residues at the N-terminus or C-terminus thereof together with the substitution, deletion and/or insertion of the amino acid residues, and is used with substantially the same meaning as the expression “PH20 variant or fragment thereof”.
Preferably, the PH20 variant according to the present invention includes amino acid residue substitution, insertion, and/or deletion at one or more positions selected from the group consisting of R39, D65 to L68, N82, T84, 1102 to 1105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463 and D461 to V463 in a variant having the amino acid sequence of SEQ ID NO: 3, and has a higher protein aggregation temperature (Tagg) than that of wild-type PH20.
The PH20 variant according to the present invention may include a mutation at 20 or fewer, preferably 17 or fewer, more preferably 15 or fewer amino acid positions in the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.
More preferably, the PH20 variant or fragment thereof according to the present invention includes at least one amino acid residue substitution selected from the group consisting of R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T, S180A, L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q268I, Q268N, Q269A, P271A, V272A, K290A, I291A, I291G, I291L, P292A, P292D, Q311A, V312A, L313A, L313P, L313M, K314A, G340Q, S341H, S341D, S341T, W342I, W342D, W342H, W342L, E343V, E343S, E343Y, E343Q, N344F, N344I, T345E, T345K, T345S, R346M, R346F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E349L, E349W, E349A, S350Q, S350I, S350D, S350T, S350E, S350N, Q352E, Q352G, Q352Y, Q352W, Q352T, A353E, A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I354R, I354W, I354L, K355Q, K355H, K355D, E356M, E356F, E356I, E356L, E356Q, E356V, E356D, Y357W, Y357F, M358V, M358R, M358Y, M358L, D359K, D359V, D359Y, D359Q, D359T, D359S, D359E, T360Y, T360R, T360L, T360D, T360S, T361M, T361E, T361H, T361L, T361D, T361I, L362A, N363M, N363E, L441A, S442A, D451A, D451S, T452A, T452D, T452H, T452K, T452G, T452P, T452M, T452F, D453A, D461R, D461A, G462A, V463Y and V463A in the variant having the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.
In the present invention, an expression described by a one-letter amino acid residue code together with numbers, such as “S341”, means the amino acid residue at each position in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
For example, “S341” means that the amino acid residue at position 341 in the amino acid sequence of SEQ ID NO: 3 is serine and “S341H” means that serine at position 341 of SEQ ID NO: 3 is substituted with histidine.
The PH20 variant or a fragment thereof according to the present invention is interpreted as including variants or fragments thereof in which an amino acid residue at a specific amino acid residue position is conservatively substituted.
As used herein, the term “conservative substitution” refers to modifications of a PH20 variant that involve the substitution of one or more amino acids with other amino acids having similar biochemical properties that do not result in loss of the biological or biochemical function of the PH20 variant.
The term “conservative amino acid substitution” refers to substitution of the amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined and are well known in the art to which the present invention pertains. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
It is found that the PH20 variant or fragments thereof of the present invention retains the activity thereof despite having conservative amino acid substitutions.
In addition, the PH20 variant or fragment thereof according to the present invention is interpreted to include PH20 variants or fragments thereof having substantially the same function and/or effect as those/that of the PH20 variant or fragment thereof according to the present invention, and having amino acid sequence homology of at least 80% or 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% to the PH20 variant or fragment thereof according to the present invention.
The PH20 variants or fragments thereof according to the present invention have increased expression levels and protein refolding rate, and thereby have higher thermal stability than mature wild-type PH20. Furthermore, the enzymatic activity of the PH20 variants was greater than or similar to that of mature wild-type PH20 despite the increase in thermal stability.
Meanwhile, although the mature wild-type PH20 variant having cleavage at the C-terminal is known to have decreased enzymatic activity, the PH20 variants according to the present invention exhibit similar or increased enzymatic activity and expression efficiency, and high protein aggregation temperatures (Tagg) due to the more rapid protein refolding and thermal stability thereof, although one or more amino acid residues at the C-terminus are cleaved and deleted, and/or 1 to 7, preferably, 1 to 5 amino acid residues at the N-terminus are cleaved and deleted.
Accordingly, the PH20 variant or fragment thereof according to the present invention is characterized in that it includes one or more amino acid mutations, preferably one or more amino acid residue substitutions, deletions and/or insertions in the variant having the amino acid sequence of SEQ ID NO: 3 or the like, and one or more amino acid residues of N-terminus and/or C-terminus are additionally deleted, but is not limited thereto.
In one embodiment, the PH20 variant or fragment thereof according to the present invention may be one in which cleavage occurs before an amino acid residue selected from the group consisting of M1 to P42 from the N-terminus, preferably before an amino acid residue L36, N37, F38, R39, A40, P41, or P42 at the N-terminus, in the amino acid sequence of SEQ ID NO: 3, so that one or more amino acid residues from the N-terminus are deleted, and/or cleavage occurs after an amino acid residue selected from the group consisting of V455 to L509, preferably after an amino acid residue selected from the group consisting of V455 to S490, most preferably after an amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 at the C-terminus, so that one or more amino acid residues at the C-terminus are deleted.
The expression “cleavage occurs before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus” means that a portion of amino acid residues immediately before the selected amino acid residue from among M1 to P42 at the N-terminus is cleaved and deleted. The expression “cleavage occurs before M1” means that no cleavage occurs at the N-terminus.
For example, the expression “cleavage occurs before an amino acid residue L36, N37, F38, R39, A40, P41, or P42” means that all amino acid residues from M1 to T35 immediately before L36, all amino acid residues from M1 to L36 immediately before N37, all amino acid residues from M1 to N37 immediately before F38, all amino acid residues from M1 to F38 immediately before R39, all amino acid residues from M1 to R39 immediately before A40, all amino acid residues from M1 to A40 immediately before P41, or all amino acid residues from M1 to P41 immediately before P42 in the amino acid sequence of SEQ ID NO: 3 according to the present invention are cleaved and removed.
In addition, the expression “cleavage occurs after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus” means that a portion of amino acid residues immediately before the selected amino acid residue from among M1 to P42 at the N-terminus is cleaved and deleted.
For example, the expression “cleavage occurs after an amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 at the C-terminus” means that an amino acid residue after the amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 in the amino acid sequence of SEQ ID NO: 3 according to the present invention is cleaved and removed.
Preferably, the novel PH20 variant or fragment thereof according to the present invention is characterized in that it includes an amino acid residue substitution, deletion or insertion at one or more positions in the variant having the amino acid sequence of SEQ ID NO: 3, a truncation before F38 at the N-terminus, and a truncation after F468 at the C-terminus.
More preferably, the novel PH20 variant or fragment thereof according to the present invention may include an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOS: 163 to 316, but is not limited thereto.
The sequences of the substituted or cleaved amino acids in the PH20 variant constructed in the specific embodiment according to the present invention are as shown in Table 6.
In addition, in the present invention, an attempt was made to increase the expression of a recombinant PH20 protein using other signal peptide of proteins highly expressed in animal cells, instead of using the original signal peptide of PH20.
Therefore, in another embodiment, the novel PH20 variant according to the present invention may be one in which the N-terminus further includes a human growth hormone signal peptide having an amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA of SEQ ID NO: 4, a human serum albumin signal peptide having an amino acid sequence MKWVTFISLLFLFSSAYS of SEQ ID NO: 5, or a human Hyal1 signal peptide having an amino acid sequence MAAHLLPICALFLTLLDMAQG of SEQ ID NO: 6 as shown in Table 3 below, instead of the signal peptide of wild-type PH20, which consists of M1 to T35, but is not limited thereto.
The expression “instead of the signal peptide of wild-type PH20, which consists of M1 to T35” means the case in which the signal peptide in the amino acid sequence of SEQ ID NO: 3 is partially or completely deleted, and thus does not perform the function thereof. In addition, the expression is meant to include the case in which a portion of the N-terminus is further deleted, for example, the case in which cleavage occurs before the N37, F38, R39, A40, P41 or P42 residue occurs so that an additional deletion of the N-terminus together with the deletion of the signal peptide of wild-type PH20 occurs.







TABLE 3







Signal peptide sequence according to 


present invention













SEQ





ID




Amino acid sequence
NO.







Human Growth 
MATGSRTSLLLAFGLLCLPWLQEGSA
4



hormone





 



Human serum 
MKWVTFISLLFLFSSAYS
5



albumin





 



Human Hyal1
MAAHLLPICALFLTLLDMAQG
6










In another aspect, the present invention is directed to a composition for treating cancer containing the novel PH20 variant or fragment thereof according to the present invention and a method for treating cancer using the same.
The cancers or carcinomas that can be treated by the novel PH20 variant or fragment thereof according to the present invention are not particularly limited, but include both solid cancers and blood cancers. The cancer may be selected from the group consisting of skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, renal cancer, esophageal cancer, biliary tract cancer, testis cancer, rectal cancer, head and neck cancer, ureteral cancer, osteosarcoma, neurocytoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and neuroglioma, but is not limited thereto. Preferably, cancers that can be treated by the composition according to the present invention may be selected from the group consisting of colorectal cancer, breast cancer, lung cancer and renal cancer, but are not limited thereto.
The composition of the present invention may be a pharmaceutical composition. The pharmaceutical composition may further include a pharmaceutically acceptable composition. The composition may contain one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, which are typically used in the preparation of drugs, but is not limited thereto. In addition, the pharmaceutical composition may further contain one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives, which are typically used in the preparation of drugs.
The pharmaceutical composition may be administered orally or parenterally. The parenteral administration is carried out by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. For oral administration, the active ingredient in the oral composition needs to be formulated into a coated dosage form or into a dosage form that can protect the active ingredient from disintegrating in the stomach, considering that peptides and proteins are digested in the stomach. Alternatively, the present composition may be administered via any device by which the active ingredient can move to the target cell of interest.
The pharmaceutical composition may be formulated in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, grains, powders, granules, tablets or capsules, and may additionally include dispersing or stabilizing agents for the purpose of formulation.
In particular, the composition for treating cancer according to the present invention may be used in combined therapy with other anticancer drugs.
An anticancer drug that can be used in combined therapy with the novel PH20 variant or fragment thereof according to the present invention is preferably a chemical anticancer drug, an antibody-based anticancer drug, a biological anticancer drug, an RNAi, or a cell therapeutic agent, but is not limited thereto.
Preferably, the anticancer drug that can be used in combined therapy with the novel PH20 variant or fragment thereof according to the present invention is preferably an immuno-oncologic agent, and more preferably an immune checkpoint inhibitor, but is not limited thereto.
In addition, the present invention is directed to a method for treating cancer using the novel PH20 variant or fragment in combination with other anticancer agents, particularly the anticancer agents described above.
In another aspect, the present invention is directed to a nucleic acid encoding the PH20 variant or fragment thereof.
The nucleic acids, as used herein, may be present in cells, in the cell lysate, or in the partially purified or substantially pure form. “Isolated” or “to be substantially pure”, when referring to nucleic acids, refer to those that have been purified and thus separated from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acids of the present invention may be DNA or RNA.
In still another aspect, the present invention is directed to a recombinant expression vector including the nucleic acid. For expression of the PH20 variant or fragment thereof according to the present invention, a DNA encoding the PH20 variant or fragment thereof can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the PH20 variant), and the DNA can be inserted into an expression vector such that it is “operatively linked” to transcriptional and translational control sequences.
As used herein, the term “operatively linked” is intended to mean that a gene encoding the PH20 variant or fragment thereof is ligated into a vector such that transcriptional and translational control sequences serve intended functions thereof of regulating the transcription and translation of the gene encoding the PH20 variant or fragment thereof. The expression vector and expression control sequences are chosen to be compatible with the expression host cell that is used. The genes encoding the PH20 are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on a fragment of the gene encoding the PH20 variant or fragment thereof and a vector, or blunt-end ligation if no restriction enzyme sites are present).
In addition, the recombinant expression vectors carry regulatory sequences that control the expression of a gene encoding the PH20 variant or fragment thereof in the host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the genes encoding the PH20 variant or fragment thereof. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the desired level of expression of the protein, etc.
In yet another aspect, the present invention is directed to a host cell including the nucleic acid or the vector. The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeasts, E. coli., and insect cells, but is not limited thereto.
Specifically, the host cell according to the present invention include prokaryotic cells such as E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, and eukaryotic cells such as lower eukaryotic cells, and higher other eukaryotic cells such as insect cells.
In addition, the host cells that can be used in the present invention may be derived from plants or mammals. Preferably, examples of the host cells include, but are not limited to, monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cells, HuT 78 cells and HEK293 cells. More preferably, CHO cells may be used.
The nucleic acid or the vector is transfected into a host cell. Transfection can be performed using various techniques that are generally used to introduce foreign nucleic acid (DNA or RNA) into prokaryotic or eukaryotic cells, for example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection. In order to express the PH20 variant or fragment thereof of the present invention, various combinations of recombinant expression vectors and host cells can be employed. The preferred expression vector for eukaryotic cells includes gene expression regulatory sequences derived from, but not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used for bacterial hosts include bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof, obtained from E. coli; a plasmid having broad host range, such as RP4; phage DNAs exemplified by various phage lambda derivatives, such as, λgt10, λgt11 and NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phage. An expression vector available for yeast cells may be a 2-μm plasmid and derivatives thereof. Expression vectors for insect cells include pVL941.
In another aspect, the present invention is directed to a method for producing a PH20 variant or fragment thereof, the method including culturing the host cell and expressing the PH20 variant or fragment thereof according to the present invention.
When a recombinant expression vector capable of expressing the PH20 variant or fragment thereof is introduced into mammalian host cells, the PH20 variant or fragment thereof can be produced by culturing the host cells for a period of time such that the PH20 variant or fragment thereof is expressed in the host cells, preferably a period of time such that the PH20 variant is secreted into the medium during culture of the host cells.
In an alternative embodiment, the expressed PH20 variant or fragment thereof can be isolated and purified from the host cells. Isolation or purification of the PH20 variant or fragment thereof can be performed by conventional isolation/purification methods (e.g., chromatography) that are used for proteins. The chromatography may include a combination of one or more selected from affinity chromatography, ion exchange chromatography, and hydrophobic chromatography, but is not limited thereto. In addition to the chromatography, a combination of filtration, ultrafiltration, salting out, dialysis, and the like may be used.
In order to confirm the industrial applicability of the enzyme, it is necessary to analyze the catalytic reaction rate of the enzyme. Types of enzymatic reactions include an enzyme reaction with an active site with fixed reactivity and an enzyme reaction with several active sites with various reactivity. It is known that the catalytic reaction rate of enzymes having an active site with fixed reactivity, such as hyaluronidase, follows the Michaelis-Menten rate formula.
The Michaelis-Menten's enzyme kinetics is premised on the assumption of an enzymatic reaction as a two-step reaction system including a reversible reaction step in which Complex [ES] of Enzyme (E)-Substrate (S) is formed and an irreversible reaction step in which the ES complex is dissociated to yield Product (P). In this case, kf, kr and kcat are the rate constants of the reaction in each direction (Alan Fersht (1977) Enzyme structure and mechanism).



 
  
   E
   +
   S
  
  
   ⇌
   
    k
    r
   
   
    k
    f
   
  
  
   
    ES
    
     ⟶
     
      k
      cat
     
    
    E
   
   +
   P
  
 



The enzymatic reaction assumes that the process of reacting the enzyme with the substrate to produce the ES complex rapidly reaches equilibrium, or may be considered to be a pseudo-steady state assuming that d[ES]/dt≈0 is satisfied by sufficiently lowering the concentration of the enzyme by performing a reaction that maintains a sufficiently high substrate concentration. Since the rate equations assuming fast equilibrium or pseudo-steady state are derived in the same manner, a pseudo-steady state in which the substrate concentration is initially higher than the enzyme concentration is assumed in most experiments.
When conditions such as “the amount of enzyme is constant before and after the reaction” and “when a chemical reaction reaches chemical equilibrium, the reaction rate at which the product is obtained is equal to the rate at which the product is decomposed again” are used under such an assumption, the reaction rate of the final product may be expressed by the following Michaelis-Menten rate formula. In this case, KM=(kr+kcat/kf, and Vmax=kcat [E]0.



 
  v
  =
  
   
    
     d
     [
     P
     ]
    
    dt
   
   =
   
    
     
      V
      max
     
     [
     S
     ]
    
    
     
      K
      M
     
     +
     
      [
      S
      ]
     
    
   
  
 



The Lineweaver-Burk equation is used to experimentally analyze the enzyme reaction rate using the Michaelis-Menten rate formula. This equation shows the relationship between the reciprocal 1/V of the experimentally measured reaction rate with the reciprocal 1/[S] of the given substrate concentration in the experiment. Statistical verification that this equation is a linear equation demonstrates that the enzyme reaction is a reaction following Michaelis-Menten's rate formula, and KM and Vmax can be calculated using this equation.
Enzymes that catalyze a chemical reaction have a transition state after binding to a substrate at an active site, and the activation energy for reaching the transition state having high energy is lowered through multiple bonds with the substrate. The equilibrium constant for reaching this transition state is proportional to kcat/KM. Here, 1/KM is an index that combines the degree to which an enzyme-substrate complex is produced by bonding the enzyme to the substrate with the degree to which the enzyme-substrate complex is maintained without being decomposed, and kcat is the equilibrium constant at which a product is obtained from the enzyme-substrate complex. Therefore, kcat/KM can be said to be an indicator of how much product can be obtained from the substrate and the enzyme, that is, the catalytic efficiency of the enzyme.
The industrial availability of hyaluronidase is proportional to the catalytic efficiency thereof. In particular, when the enzyme is injected subcutaneously along with a polymeric pharmacologically active substance such as a monoclonal antibody, the catalytic efficiency of hyaluronidase plays an important role. In the case where the variant according to the present invention has higher kcat/KM than the wild-type PH20, when the hyaluronidase combined with the polymeric pharmacologically active substance is administered subcutaneously, hyaluronic acid present therein is rapidly decomposed and thus a superior effect of rapidly dispersing the pharmacologically active substance can be obtained. In addition, when the variant according to the present invention has a larger kcat than the wild-type PH20, the maximum reaction rate Vmax increases at the same enzyme concentration, thereby providing excellent effects of decomposing a greater amount of hyaluronic acid during the same period of time and dispersing the pharmacologically active substance in a wider region.
Therefore, in order to confirm the enzymatic properties of the PH20 variant according to the present invention, the enzyme reaction rate of each variant was analyzed, and Vmax (maximum enzyme reaction rate), KM (substrate concentration under 50% Vmax condition), kcat (substrate conversion rate), and kcat/KM (enzyme catalyst efficiency) thereof were compared in Example 4. The results described above demonstrate that the PH20 variant according to the present invention is superior to wild-type PH20.
EXAMPLE
Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.
Example 1. Construction of PH20 Variants
For construction of PH20 variants, cDNA (clone ID: hMU002604) of wild-type PH20 was purchased from the Korean Human Gene Bank. Wild-type PH20 encodes amino acids from L36 to S490. The PH20 gene was amplified by polymerase chain reaction (hereinafter referred to as PCR) and inserted into the XhoI and NotI restriction enzyme sites of a pcDNA3.4-TOPO vector. For expression in EXPICHO™ cells, the signal peptide of human growth hormone, human serum hormone or human Hyal1 was used as a signal peptide instead of the original signal peptide of PH20. For protein purification using a HisTrap column, the DNA sequence of a His-tag was located at the 3′-end of the PH20 cDNA. The amino acid substitution of PH20 variants was performed using PCR, and the amino acid substitution was confirmed through DNA sequencing.
The list of primers used in cloning of the PH20 variants are summarized in Table 4 below, and the specific sequences of the primers are summarized in Table 5 below.







TABLE 4







List of primers used in cloning of PH 20


variants according to present invention









Primer










Clone
1
2
3





cB4205
ALB-SP-Xho
B4-hy2
SPAM1-6H-not


cB4206
ALB-SP-Xho
B4-hy3
SPAM1-6H-not


cB4207
ALB-SP-Xho
B4-hy4
SPAM1-6H-not


cB4213-m63
opB4-Xho-hSA
op-F468-6H-not
—


cB4213-m64
opB4-Xho-hSA
op-Q347-m64
op-F468-6H-not


cB4213-m65
op-Xho-hSA-L
op-Q348-m65
op-F468-R


cB4213-m66
op-Xho-hSA-L
op-Q350-m66
op-F468-R


cB4213-m67
opB4-Xho-hSA
op-Q355-m67
op-F468-6H-not


cB4213-m69
op-Xho-hSA-L
op-V358-m69
op-F468-6H-not


cB4213-m70
op-Xho-hSA-L
op-A362-m70
op-F468-6H-not


cB4213-m71
opB4-Xho-hSA
op-V343-m71
op-F468-6H-not


cB4213-m72
opB4-Xho-hSA
op-F344-m72
op-F468-6H-not


cB4213-m73
op-Xho-hSA-L
op-K359-mega-
op-F468-6H-not




NL73



cB4213-m74
op-Xho-hSA-L
op-Y360-m74
op-F468-6H-not


cB4213-m75
opB4-Xho-hSA
op-M361-m75
op-F468-6H-not


cB4213-m76
opB4-Xho-hSA
op-E352-m76
op-F468-6H-not


cB4213-m77
opB4-Xho-hSA
op-M363-m77
op-F468-6H-not


cB4213-m78
opB4-Xho-hSA
op-N84-m78
op-F468-6H-not


cB4213-m79
opB4-Xho-hSA
op-K166-m79
op-F468-6H-not


cB4213-m82
op-Xho-hSA-L
op-354E-m82
op-F468-6H-not


cB4213-m83
op-Xho-hSA-L
op-354Q-m83
op-F468-6H-not


cB4213-m84
op-Xho-hSA-L
op-354S-m84
op-F468-6H-not


cB4213-m85
op-Xho-hSA-L
op-354V-m85
op-F468-6H-not


cB4213-m86
op-Xho-hSA-L
op-354A-m86
op-F468-6H-not


cB4213-m88
op-Xho-hSA-L
op-354N-m88
op-F468-6H-not


cB4213-m89
op-Xho-hSA-L
op-354T-m89
op-F468-6H-not


cB4213-m90
op-Xho-hSA-L
op-356M-m90
op-F468-6H-not


cB4213-m91
op-Xho-hSA-L
op-356F-m91
op-F468-6H-not


cB4213-m92
op-Xho-hSA-L
op-356I-m92
op-F468-6H-not


cB4213-m93
op-Xho-hSA-L
op-356L-m93
op-F468-6H-not


cB4213-m94
op-Xho-hSA-L
op-356Q-m94
op-F468-6H-not


cB4213-m95
op-Xho-hSA-L
op-356V-m95
op-F468-6H-not


cB4213-m96
op-Xho-hSA-L
op-343V_364M-
op-F468-6H-not




m96



cB4213-m97
op-Xho-hSA-L
op-340Q-m97
op-F468-6H-not


cB4213-m98
op-Xho-hSA-L
op-341H-m98
op-F468-6H-not


cB4213-m99
op-Xho-hSA-L
op-342I-m99
op-F468-6H-not


cB4213-m100
op-Xho-hSA-L
op-343Y-m100
op-F468-6H-not


cB4213-m101
op-Xho-hSA-L
op-345E-m101
op-F468-6H-not


cB4213-m102
op-Xho-hSA-L
op-346F-m102
op-F468-6H-not


cB4213-m103
op-Xho-hSA-L
op-347E-m103
op-F468-6H-not


cB4213-m104
op-Xho-hSA-L
op-349L-m104
op-F468-6H-not


cB4213-m105
op-Xho-hSA-L
op-350I-m105
op-F468-6H-not


cB4213-m106
op-Xho-hSA-L
op-352G-m106
op-F468-6H-not


cB4213-m107
op-Xho-hSA-L
op-354R-m107
op-F468-6H-not


cB4213-m110
op-Xho-hSA-L
op-358R-m110
op-F468-6H-not


cB4213-m111
op-Xho-hSA-L
op-359V-m111
op-F468-6H-not


cB4213-m112
op-Xho-hSA-L
op-360R-m112
op-F468-6H-not


cB4213-m114
op-Xho-hSA-L
op-345K-m114
op-F468-6H-not


cB4213-m115
op-Xho-hSA-L
op-346L-m115
op-F468-6H-not


cB4213-m116
op-Xho-hSA-L
op-347V-m116
op-F468-6H-not


cB4213-m117
op-Xho-hSA-L
op-349W-m117
op-F468-6H-not


cB4213-m118
op-Xho-hSA-L
op-354W-m118
op-F468-6H-not


cB4213-m121
op-Xho-hSA-L
op-359Y-m121
op-F468-6H-not


cB4213-m125
op-Xho-hSA-L
op-347W-m125
op-F468-6H-not


cB4213-m126
op-Xho-hSA-L
op-357W-m126
op-F468-6H-not


cB4213-m130
op-Xho-hSA-L
op-342D-m130
op-F468-6H-not


cB4213-m131
op-Xho-hSA-L
op-343Q-m131
op-F468-6H-not


cB4213-m132
op-Xho-hSA-L
op-347H-m132
op-F468-6H-not


cB4213-m133
op-Xho-hSA-L
op-348F-m133
op-F468-6H-not


cB4213-m134
op-Xho-hSA-L
op-350D-m134
op-F468-6H-not


cB4213-m135
op-Xho-hSA-L
op-352Y-m135
op-F468-6H-not


cB4213-m136
op-Xho-hSA-L
op-353E-m136
op-F468-6H-not


cB4213-m138
op-Xho-hSA-L
op-358Y-m138
op-F468-6H-not


cB4213-m139
op-Xho-hSA-L
op-359Q-m139
op-F468-6H-not


cB4213-m140
op-Xho-hSA-L
op-360L-m140
op-F468-6H-not


cB4213-m141
op-Xho-hSA-L
op-361E-m141
op-F468-6H-not


cB4213-m142
op-Xho-hSA-L
op-363E-m142
op-F468-6H-not


cB4213-m143
op-Xho-hSA-L
op-342H-m143
op-F468-6H-not


cB4213-m144
op-Xho-hSA-L
op-348D-m144
op-F468-6H-not


cB4213-m145
op-Xho-hSA-L
op-361H-m145
op-F468-6H-not


cB4213-m146
opB4-Xho-hSA
Op-R39-m146-R
op-F468-6H-not


cB4213-m147
opB4-Xho-hSA
Op-A40-m147-R
op-F468-6H-not


cB4213-m149
opB4-Xho-hSA
op-D456-6H-not
—


cB4213-m150
op-Xho-hSA-L
op-350Q360R-m150
op-F468-6H-not


cB4213-m152
opB4-Xho-hSA
Op-m152-D65A-R
op-F468-6H-not


cB4213-m153
opB4-Xho-hSA
Op-m153-E66A-R
op-F468-6H-not


cB4213-m154
opB4-Xho-hSA
Op-m154-P67A-R
op-F468-6H-not


cB4213-m155
opB4-Xho-hSA
Op-m155-L68A-R
op-F468-6H-not


cB4213-m156
op-Xho-hSA-L
op-311A-m156
op-F468-6H-not


cB4213-m157
op-Xho-hSA-L
op-312A-m157
op-F468-6H-not


cB4213-m158
op-Xho-hSA-L
op-313A-m158
op-F468-6H-not


cB4213-m159
op-Xho-hSA-L
op-314A-m159
op-F468-6H-not


cB4213-m160
op-Xho-hSA-L
N266A-m160
op-F468-6H-not


cB4213-m161
op-Xho-hSA-L
T267A-m161
op-F468-6H-not


cB4213-m162
op-Xho-hSA-L
Q268A-m162
op-F468-6H-not


cB4213-m163
op-Xho-hSA-L
Q269A-m163
op-F468-6H-not


cB4213-m164
op-Xho-hSA-L
P271A-m164
op-F468-6H-not


cB4213-m165
op-Xho-hSA-L
V272A-m165
op-F468-6H-not


cB4213-m166
opB4-Xho-hSA
Op-m166-I102A-R
op-F468-6H-not


cB4213-m167
opB4-Xho-hSA
Op-m167-D103A-R
op-F468-6H-not


cB4213-m168
opB4-Xho-hSA
Op-m168-S104A-R
op-F468-6H-not


cB4213-m169
opB4-Xho-hSA
Op-m169-I105A-R
op-F468-6H-not


cB4213-m170
op-Xho-hSA-L
op-m170-T132A-R
op-F468-6H-not


cB4213-m171
op-Xho-hSA-L
op-m171-F133A-R
op-F468-6H-not


cB4213-m172
op-Xho-hSA-L
op-m172-Y134A-R
op-F468-6H-not


cB4213-m173
op-Xho-hSA-L
V241A-m173
op-F468-6H-not


cB4213-m174
op-Xho-hSA-L
E242A-m174
op-F468-6H-not


cB4213-m175
op-Xho-hSA-L
I243A-m175
op-F468-6H-not


cB4213-m176
op-Xho-hSA-L
K244A-m176
op-F468-6H-not


cB4213-m177
opB4-Xho-hSA
Op-m177-L179A-R
op-F468-6H-not


cB4213-m178
opB4-Xho-hSA
Op-m178-S180A-R
op-F468-6H-not


cB4213-m179
opB4-Xho-hSA
Op-m179-L181A-R
op-F468-6H-not


cB4213-m180
opB4-Xho-hSA
Op-m180-T182A-R
op-F468-6H-not


cB4213-m181
opB4-Xho-hSA
Op-m181-T185A-R
op-F468-6H-not


cB4213-m182
opB4-Xho-hSA
Op-m182-E186A-R
op-F468-6H-not


cB4213-m183
opB4-Xho-hSA
Op-m183-K187A-R
op-F468-6H-not


cB4213-m184
op-Xho-hSA-L
op-K290A-m184
op-F468-6H-not


cB4213-m185
op-Xho-hSA-L
op-I291A-m185
op-F468-6H-not


cB4213-m186
op-Xho-hSA-L
op-P292A-m186
op-F468-6H-not


cB4213-m190
op-Xho-hSA-L
L441A-m190
op-F468-6H-not


cB4213-m191
op-Xho-hSA-L
S442A-m191
op-F468-6H-not


cB4213-m192
opB4-Xho-hSA
op-D451A-m192
op-F468-6H-not


cB4213-m193
opB4-Xho-hSA
op-T452A-m193
op-F468-6H-not


cB4213-m194
op-Xho-hSA-L
op-D453A-m194
op-F468-6H-not


cB4213-m195
op-Xho-hSA-L
op-D461A-6H-not
op-F468-6H-not


cB4213-m196
op-Xho-hSA-L
op-G462A-6H-not
op-F468-6H-not


cB4213-m197
op-Xho-hSA-L
op-V463A-6H-not
op-F468-6H-not


cB4213-m198
op-Xho-hSA-L
op-N82A-m198-R
op-F468-6H-not


cB4213-m199
op-Xho-hSA-L
op-N166A-m199-R
op-F468-6H-not


cB4213-m203
op-Xho-hSA-L
Op-S104N-m203-R
op-F468-6H-not


cB4213-m204
op-Xho-hSA-L
Op-I105Q-m204-R
op-F468-6H-not


cB4213-m205
op-Xho-hSA-L
op-Q268D-m205-F
op-F468-6H-not


cB4213-m208
op-Xho-hSA-L
op-Q268I-m208-F
op-F468-6H-not


cB4213-m210
op-Xho-hSA-L
op-291G-m210-F
op-F468-6H-not


cB4213-m211
op-Xho-hSA-L
op-292D-m211-F
op-F468-6H-not


cB4213-m212
op-Xho-hSA-L
op-T452D-m212
op-F468-6H-not


cB4213-m213
op-Xho-hSA-L
op-T452H-m213
op-F468-6H-not


cB4213-m214
op-Xho-hSA-L
op-T452K-m214
op-F468-6H-not


cB4213-m216
op-Xho-hSA-L
Op-T452G-m216
op-F468-6H-not


cB4213-m217
op-Xho-hSA-L
Op-T452P-m217
op-F468-6H-not


cB4213-m218
op-Xho-hSA-L
op-T452M-m218
op-F468-6H-not


cB4213-m219
op-Xho-hSA-L
op-T452F-m219
op-F468-6H-not


cB4213-m220
op-Xho-hSA-L
op-D461R-6H-not-
op-F468-6H-not




m220



cB4213-m231
op-Xho-hSA-L
op-V463Y-6H-not-
—




m231



cB4213-m232
op-Xho-hSA-L
op-S180T-R-m232
op-F468-6H-not


cB4213-m233
op-Xho-hSA-L
op-D451S-F-m233
op-F468-6H-not


cB4213-m234
op-Xho-hSA-L
op-L313P-m234-F
op-F468-6H-not


cB4213-m235
op-Xho-hSA-L
op-L313M-m235-F
op-F468-6H-not


cB4213-m243
op-Xho-hSA-L
op-L179S-m243-R
op-F468-6H-not


cB4213-m245
op-Xho-hSA-L
op-L179I-m245-R
op-F468-6H-not


cB4213-m246
op-Xho-hSA-L
op-L179F-m246-R
op-F468-6H-not


cB4213-m254
op-Xho-hSA-L
FQQ-Mega-m254
op-F468-6H-not


cB4213-m261
op-Xho-hSA-L
op-Q268N-m259-m
op-F468-6H-not


cB4213-m262
op-Xho-hSA-L
B4-124-R
op-F468-6H-not


cB4213-m263
op-Xho-hSA-L
B4-124-R
op-F468-6H-not


cB4213-m266
op-Xho-hSA-L
op-L181M, E186D-
op-F468-6H-not




m266



cB4213-m268
opB4-Xho-hSA
op-Q268A-m268-m
op-F468-6H-not


cB4213-m271
opB4-Xho-hSA
op-344I, 348M-
op-F468-6H-not




m271



cB4213-m275
op-Xho-hSA-L
op-DLSS-m275
op-F468-6H-not


cB4213-m276
op-Xho-hSA-L
op-DLS-m276
op-F468-6H-not


cB4213-m279
opB4-Xho-hSA
Op-K348M-m279
op-F468-6H-not


cB4213-m280
opB4-Xho-hSA
Op-N344I K348M-
op-F468-6H-not




m280



cB4213-m287
op-Xho-hSA-L
Q268A-m162
op-F468-6H-not


cB4213-m288
opB4-Xho-hSA
Q268A-m162
op-F468-6H-not
















TABLE 5







Primer sequences used for cloning of PH20


variants










SEQ




ID



Primer
NO
Nucleotide sequence (5′->3′)





B4-hy2
  7
ATA TGG GGA ACC CTC AGT ATA ACT ACA AGC ACT GAG




ACC TGC CAA TAT CTG AAG GAT TAC CTG ACC AGA CTG




CTG AAT CCT TAC ATA ATC AAC


 


B4-hy3
  8
ATA TGG GGA ACC CTC AGT ATA TCC AGC AGT GAG GAA




GAA TGC TGG CAT TTG CAC GAT TAC CTG GTA GAC ACA




CTG AAT CCT TAC ATA ATC AAC


 


B4-hy4
  9
ATA TGG GGA ACC CTC AGT ATA ACC GCA TCT AAG GCA




AAC TGC ACA AAA GTA AAA CAA TTC GTC TCC AGT GAT




CTG AAT CCT TAC ATA ATC AAC


 


ALB-SP-Xho
 10
GAA TAT CTC GAG GCC ACC ATG AAG TGG GTT ACA


 


SPAM1-6H-not
 11
CTA ATT GCG GCC GCT CAT TAG TGG TGA TGG TGA TGA




TGG AAG AAA CCA ATT CTG C


 


op-F468-R
 12
AAT TAG GCG GCC GCC TAT TAA AAG GCG TCG ATG CAC




ACG CCA TC


 


op-F468-6H-not
 13
CTC TAA TTG CGG CCG CTC ATT AGT GGT GAT GGT GAT




GAT GAA AGG CGT CGA TGC ACA CGC CAT C


 


op-Xho-hSA-L
 14
AAT TAG AGC TCG AGG CCA CCA TGA AAT GGG TGA CCT




TTA TCT CC


 


opB4-Xho-hSA
 15
CAG ATT CTC GAG GCC ACC ATG AAA TGG G


 


op-Q347-m64
 16
ATC TGG GGC TCC TGG GAG AAC ACC AGG CAG AAG GAG




AGC TGC CAG GCC ATC


 


op-Q348-m65
 17
ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC CAG GAG




AGC TGC CAG GCC ATC AAG


 


op-Q350-m66
 18
AGA ACA CCA GGA CCA AGG AGC AAT GCC AGG CCA TCA




AGG AGT AC


 


op-Q355-m67
 19
AGG AGA GCT GCC AGG CCA TCC AGG AGT ACA TGG ACA




CAA CCC TG


 


op-V358-m69
 20
AGC TGC CAG GCC ATC AAG GAG TAC GTG GAC ACA ACC




CTG AAC CCT TAT ATC


 


op-A362-m70
 21
AGG AGT ACA TGG ACA CAA CCG CGA ACC CTT ATA TCA




TCA ATG


 


op-V343-m71
 22
ATC GTG ATC TGG GGC TCC TGG GTG AAC ACC AGG ACC




AAG GAG AG


 


op-F344-m72
 23
ATC TGG GGC TCC TGG GAG TTC ACC AGG ACC AAG GAG




AGC TG


 


op-K359-mega-
 24
AGC TGC CAG GCC ATC AAG GAG TAC ATG AAA ACA ACC


NL73

CTG AAC CCT TAT ATC


 


op-Y360-m74
 25
ATC AAG GAG TAC ATG GAC TAC ACC CTG AAC CCT TAT




ATC ATC


 


op-M361-m75
 26
ATC AAG GAG TAC ATG GAC ACA ATG CTG AAC CCT TAT




ATC ATC


 


op-E352-m76
 27
ACC AGG ACC AAG GAG AGC TGC GAG GCC ATC AAG GAG




TAC ATG G


 


op-M363-m77
 28
AGT ACA TGG ACA CAA CCC TGA TGC CTT ATA TCA TCA




ATG TGA C


 


op-N84-m78
 29
TAG AAG ATT GTC ACG CCC TGG CCG TTG GCA TTG ATC




CGA GGA GAG C


 


op-K166-m79
 30
TGC ACC AGC TCG ATG GAC CGT TTC TTA TAC ACG TCC




TTA GGC TTC


 


op-354E-m82
 31
ACC AAG GAG AGC TGC CAG GCC GAA AAG GAG TAC ATG




GAC ACA ACC


 


op-354Q-m83
 32
ACC AAG GAG AGC TGC CAG GCC CAA AAG GAG TAC ATG




GAC ACA ACC


 


op-354S-m84
 33
ACC AAG GAG AGC TGC CAG GCC TCT AAG GAG TAC ATG




GAC ACA ACC


 


op-354V-m85
 34
ACC AAG GAG AGC TGC CAG GCC GTC AAG GAG TAC ATG




GAC ACA ACC


 


op-354A-m86
 35
ACC AAG GAG AGC TGC CAG GCC GCG AAG GAG TAC ATG




GAC ACA ACC


 


op-354N-m88
 36
ACC AAG GAG AGC TGC CAG GCC AAC AAG GAG TAC ATG




GAC ACA ACC


 


op-354T-m89
 37
ACC AAG GAG AGC TGC CAG GCC ACC AAG GAG TAC ATG




GAC ACA ACC


 


op-356M-m90
 38
AAG GAG AGC TGC CAG GCC ATC AAG ATG TAC ATG GAC




ACA ACC CTG AAC


 


op-356F-m91
 39
AAG GAG AGC TGC CAG GCC ATC AAG TTC TAC ATG GAC




ACA ACC CTG AAC


 


op-356I-m92
 40
AAG GAG AGC TGC CAG GCC ATC AAG ATA TAC ATG GAC




ACA ACC CTG AAC


 


op-356L-m93
 41
AAG GAG AGC TGC CAG GCC ATC AAG TTG TAC ATG GAC




ACA ACC CTG AAC


 


op-356Q-m94
 42
AAG GAG AGC TGC CAG GCC ATC AAG CAG TAC ATG GAC




ACA ACC CTG AAC


 


op-356V-m95
 43
AAG GAG AGC TGC CAG GCC ATC AAG GTA TAC ATG GAC




ACA ACC CTG AAC


 


op-
 44
ATC GTG ATC TGG GGC TCC TGG GTG AAC ACC AGG ACC


343V_364M-

AAG GAG AGC TGC CAG GCC ATC AAG GAG TAC ATG GAC


m96

ACA ATG CTG AAC CCT TAT ATC ATC


 


op-340Q-m97
 45
AGC TAG CGG CAT CGT GAT CTG GCA ATC CTG GGA GAA




CAC CAG GAC C


 


op-341H-m98
 46
AGC GGC ATC GTG ATC TGG GGC CAC TGG GAG AAC ACC




AGG ACC AAG


 


op-342I-m99
 47
AGC GGC ATC GTG ATC TGG GGC TCC ATT GAG AAC ACC




AGG ACC AAG GAG


 


op-343Y-m100
 48
ATC GTG ATC TGG GGC TCC TGG TAT AAC ACC AGG ACC




AAG GAG AG


 


op-345E-m101
 49
ATC GTG ATC TGG GGC TCC TGG GAG AAC GAA AGG ACC




AAG GAG AGC TGC C


 


op-346F-m102
 50
ATC TGG GGC TCC TGG GAG AAC ACC TTC ACC AAG GAG




AGC TGC CAG GC


 


op-347E-m103
 51
ATC TGG GGC TCC TGG GAG AAC ACC AGG GAA AAG GAG




AGC TGC CAG GCC ATC


 


op-349L-m104
 52
ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC AAG TTG




AGC TGC CAG GCC ATC AAG G


 


op-350I-m105
 53
AGA ACA CCA GGA CCA AGG AGA TCT GCC AGG CCA TCA




AGG AG


 


op-352G-m106
 54
ACC AGG ACC AAG GAG AGC TGC GGG GCC ATC AAG GAG




TAC ATG GAC


 


op-354R-m107
 55
ACC AAG GAG AGC TGC CAG GCC AGA AAG GAG TAC ATG




GAC ACA AC


 


op-358R-m110
 56
AGC TGC CAG GCC ATC AAG GAG TAC CGG GAC ACA ACC




CTG AAC CCT TAT ATC


 


op-359V-m111
 57
AGG CCA TCA AGG AGT ACA TGG TCA CAA CCC TGA ACC




CTT ATA TC


 


op-360R-m112
 58
AGG CCA TCA AGG AGT ACA TGG ACA GAA CCC TGA ACC




CTT ATA TCA TC


 


op-345K-m114
 59
ATC TGG GGC TCC TGG GAG AAC AAG AGG ACC AAG GAG




AGC TGC CAG


 


op-346L-m115
 60
ATC TGG GGC TCC TGG GAG AAC ACC CTG ACC AAG GAG




AGC TGC CAG GC


 


op-347V-m116
 61
ATC TGG GGC TCC TGG GAG AAC ACC AGG GTC AAG GAG




AGC TGC CAG GCC ATC


 


op-349W-m117
 62
ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC AAG TGG




AGC TGC CAG GCC ATC AAG GAG


 


op-354W-m118
 63
ACC AAG GAG AGC TGC CAG GCC TGG AAG GAG TAC ATG




GAC ACA AC


 


op-359Y-m121
 64
AGG CCA TCA AGG AGT ACA TGT ACA CAA CCC TGA ACC




CTT ATA TC


 


op-347W-m125
 65
ATC TGG GGC TCC TGG GAG AAC ACC AGG TGG AAG GAG




AGC TGC CAG GCC ATC


 


op-357W-m126
 66
AGC TGC CAG GCC ATC AAG GAG TGG ATG GAC ACA ACC




CTG AAC CC


 


op-342D-m130
 67
AGC GGC ATC GTG ATC TGG GGC TCC GAC GAG AAC ACC




AGG ACC AAG GAG


 


op-343Q-m131
 68
ATC GTG ATC TGG GGC TCC TGG CAG AAC ACC AGG ACC




AAG GAG AGC


 


op-347H-m132
 69
ATC TGG GGC TCC TGG GAG AAC ACC AGG CAC AAG GAG




AGC TGC CAG GCC ATC


 


op-348F-m133
 70
ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC TTC GAG




AGC TGC CAG GCC ATC AAG


 


op-350D-m134
 71
AGA ACA CCA GGA CCA AGG AGG ACT GCC AGG CCA TCA




AGG AGT AC


 


op-352Y-m135
 72
ACC AGG ACC AAG GAG AGC TGC TAC GCC ATC AAG GAG




TAC ATG GAC AC


 


op-353E-m136
 73
AGG ACC AAG GAG AGC TGC CAG GAA ATC AAG GAG TAC




ATG GAC AC


 


op-358Y-m138
 74
AGC TGC CAG GCC ATC AAG GAG TAC TAC GAC ACA ACC




CTG AAC CCT TAT ATC


 


op-359Q-m139
 75
AGG CCA TCA AGG AGT ACA TGC AGA CAA CCC TGA ACC




CTT ATA TC


 


op-360L-m140
 76
AGG CCA TCA AGG AGT ACA TGG ACC TAA CCC TGA ACC




CTT ATA TCA TC


 


op-361E-m141
 77
ATC AAG GAG TAC ATG GAC ACA GAG CTG AAC CCT TAT




ATC ATC AAT G


 


op-363E-m142
 78
AGT ACA TGG ACA CAA CCC TGG AGC CTT ATA TCA TCA




ATG TGA C


 


op-342H-m143
 79
AGC GGC ATC GTG ATC TGG GGC TCC CAT GAG AAC ACC




AGG ACC AAG GAG


 


op-348D-m144
 80
ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC GAC GAG




AGC TGC CAG GCC ATC AAG


 


op-361H-m145
 81
ATC AAG GAG TAC ATG GAC ACA CAC CTG AAC CCT TAT




ATC ATC AAT G


 


Op-R39-m146-
 82
TTT GGA ATC ACA GGA GGA GCC CGA GAG TAT GCG GAG


R

CTA AAC AG


 


Op-A40-m147-
 83
TTT GGA ATC ACA GGA GGA GCA GAG TAT GCG GAG CTA


R

AAC AG


 


op-D456-6H-
 84
CTC TAA TTG CGG CCG CCT ATT AGT GGT GAT GGT GAT


not

GAT GGT CCA CGG CAT CTG TGT CCT TC


 


op-350Q360R-
 85
AGA ACA CCA GGA CCA AGG AGC AGT GCC AGG CCA TCA


m150

AGG AGT ACA TGG ACC GAA CCC TGA ACC CTT ATA TCA TC


 


Op-m152-
 86
TAA AAG AGA ACA GGC TCA TAT CCA GGG GCT CGG CAA


D65A-R

ACT TGC CCA GGC AGA ACT C


 


Op-m153-
 87
TAA AAG AGA ACA GGC TCA TAT CCA GGG GCG CGT CAA


E66A-R

ACT TGC CCA GGC AGA AC


 


Op-m154-
 88
TAA AAG AGA ACA GGC TCA TAT CCA GGG CCT CGT CAA


P67A-R

ACT TGC CCA GGC


 


Op-m155-
 89
TAA AAG AGA ACA GGC TCA TAT CCG CGG GCT CGT CAA


L68A-R

ACT TGC CCA G


 


op-311A-m156
 90
ACC AGG ATC GTG TTT ACA GAC GCG GTG CTG AAG TTC




CTG TCC


 


op-312A-m157
 91
AGG ATC GTG TTT ACA GAC CAG GCG CTG AAG TTC CTG




TCC CAG


 


op-313A-m158
 92
ATC GTG TTT ACA GAC CAG GTG GCG AAG TTC CTG TCC




CAG GAT


 


op-314A-m159
 93
ATC GTG TTT ACA GAC CAG GTG CTG GCG TTC CTG TCC




CAG GAT GAG


 


N266A-m160
 94
GCC CTG TAC CCT AGC ATC TAT CTG GCC ACC CAG CAG




AGC CCA GTG GC


 


T267A-m161
 95
CTG TAC CCT AGC ATC TAT CTG AAC GCC CAG CAG AGC




CCA GTG GCC GCT AC


 


Q268A-m162
 96
TAC CCT AGC ATC TAT CTG AAC ACC GCG CAG AGC CCA




GTG GCC GCT ACA CTG


 


Q269A-m163
 97
TAC CCT AGC ATC TAT CTG AAC ACC CAG GCG AGC CCA




GTG GCC GCT ACA CTG TAT G


 


P271A-m164
 98
AGC ATC TAT CTG AAC ACC CAG CAG AGC GCA GTG GCC




GCT ACA CTG TAT GTG AGG


 


V272A-m165
 99
TAT CTG AAC ACC CAG CAG AGC CCA GCG GCC GCT ACA




CTG TAT GTG AGG


 


Op-m166-
100
TGT CAC TCC GGT GAT AGA ATC GGC ATA TGG ATA GTA


I102A-R

GCC CAG TCT G


 


Op-m167-
101
TCA CTG TCA CTC CGG TGA TAG AAG CGA TAT ATG GAT


D103A-R

AGT AGC CCA G


 


Op-m168-
102
TCC GTT CAC TGT CAC TCC GGT GAT AGC ATC GAT ATA


S104A-R

TGG ATA GTA GCC CAG


 


Op-m169-
103
TCC GTT CAC TGT CAC TCC GGT GGC AGA ATC GAT ATA


I105A-R

TGG ATA GTA GC


 


op-m170-
104
TTG TCC ACT GGC ATG TAG AAG GCG ATG TCC TTC TTA


T132A-R

GCC TTA TC


 


op-m171-
105
TGC CCA GAT TGT CCA CTG GCA TGT AGG CGG TGA TGT


F133A-R

CCT TCT TAG CCT TAT C


 


op-m172-
106
TGC CCA GAT TGT CCA CTG GCA TGG CGA AGG TGA TGT


Y134A-R

CCT TCT TAG


 


V241A-m173
107
AGA TCG TCG TTC CTC TTG ATC TCC GCA TTG AAA CAG




GAG CCG TTG TAG CC


 


E242A-m174
108
GAC AGA TCG TCG TTC CTC TTG ATC GCC ACA TTG AAA




CAG GAG CCG TTG TAG CC


 


I243A-m175
109
AGC CAA GAC AGA TCG TCG TTC CTC TTG GCC TCC ACA




TTG AAA CAG GAG CCG TTG


 


K244A-m176
110
AGC CAA GAC AGA TCG TCG TTC CTC GCG ATC TCC ACA




TTG AAA CAG GAG CCG


 


Op-m177-
111
TCT GTG GCC TCG GTC AGG CTC GCC TGC ACG TTC TGC


L179A-R

TGC TGC AC


 


Op-m178-
112
TTC TCT GTG GCC TCG GTC AGG GCC AGC TGC ACG TTC


S180A-R

TGC TGC TG


 


Op-m179-
113
TAG CCT TCT CTG TGG CCT CGG TCG CGC TCA GCT GCA


L181A-R

CGT TCT GCT G


 


Op-m180-
114
TTA GCC TTC TCT GTG GCC TCG GCC AGG CTC AGC TGC


T182A-R

ACG TTC TG


 


Op-m181-
115
TCG AAC TCC TGC TTA GCC TTC TCT GCG GCC TCG GTC


T185A-R

AGG CTC AGC TG


 


Op-m182-
116
TCG AAC TCC TGC TTA GCC TTC GCT GTG GCC TCG GTC


E186A-R

AGG CTC AG


 


Op-m183-
117
TTC TCG AAC TCC TGC TTA GCC GCC TCT GTG GCC TCG GTC


K187A-R

AGG C


 


op-K290A-
118
AGA GAG GCT ATC CGC GTG TCT GCG ATC CCC GAC GCC


m184

AAG TCC CCA C


 


op-I291A-m185
119
AGG CTA TCC GCG TGT CTA AGG CCC CCG ACG CCA AGT




CCC CAC TG


 


op-P292A-
120
AGG CTA TCC GCG TGT CTA AGA TCG CCG ACG CCA AGT


m186

CCC CAC TGC CC


 


L441A-m190
121
AGT TTT ACT GCT CTT GTT ATT CCA CCG CGA GCT GTA




AGG AGA AGG CTG ATG


 


S442A-m191
122
ACT GCT CTT GTT ATT CCA CCC TGG CCT GTA AGG AGA




AGG CTG ATG TG


 


op-D451A-
123
AAG GAG AAG GCT GAT GTG AAG GCC ACA GAT GCC GTG


m192

GAC GTG TGC


 


op-T452A-
124
AAG GAG AAG GCT GAT GTG AAG GAC GCA GAT GCC GTG


m193

GAC GTG TGC ATC


 


op-D453A-
125
AAG GCT GAT GTG AAG GAC ACA GCT GCC GTG GAC GTG


m194

TGC ATC G


 


op-D461A-6H-
126
ATA TTC GCG GCC GCC TAT TAG TGG TGA TGG TGA TGA


not

TGA AAG GCG TCG ATG CAC ACG CCA GCA GCG ATG CAC




ACG TCC ACG


 


op-G462A-6H-
127
ATA TTC GCG GCC GCC TAT TAG TGG TGA TGG TGA TGA


not

TGA AAG GCG TCG ATG CAC ACG GCA TCA GCG ATG CAC




ACG TCC AC


 


op-V463A-6H-
128
ATA TTC GCG GCC GCC TAT TAG TGG TGA TGG TGA TGA


not

TGA AAG GCG TCG ATG CAC GCG CCA TCA GCG ATG CAC




ACG


 


op-N82A-
129
TGT CAC GCC CTG GCC GGT GGC AGC GAT CCG AGG AGA


m198-R

GCC GAT AAA AG


 


op-N166A-
130
TGC ACC AGC TCG ATG GAC CGA GCC TTA TAC ACG TCC


m199-R

TTA GGC TTC


 


Op-S104N-
131
TTC ACT GTC ACT CCG GTG ATA TTA TCG ATA TAT GGA


m203-R

TAG TAG CC


 


Op-I105Q-
132
TCC GTT CAC TGT CAC TCC GGT CTG AGA ATC GAT ATA


m204-R

TGG ATA GTA GC


 


op-Q268D-
133
ACC CTA GCA TCT ATC TGA ACA CCG ATC AGA GCC CAG


m205-F

TGG CCG CTA C


 


op-Q268I-
134
ACC CTA GCA TCT ATC TGA ACA CCA TCC AGA GCC CAG


m208-F

TGG CCG CTA C


 


op-291G-m210-
135
AGG CTA TCC GCG TGT CTA AGG GCC CCG ACG CCA AGT


F

CCC CAC


 


op-292D-m211-
136
ATC CGC GTG TCT AAG ATC GAC GAC GCC AAG TCC CCA


F

CTG C


 


op-T452D-
137
AGA AGG CTG ATG TGA AGG ACG ACG ATG CCG TGG ACG


m212

TGT G


 


op-T452H-
138
AGA AGG CTG ATG TGA AGG ACC ACG ATG CCG TGG ACG


m213

TGT G


 


op-T452K-
139
AGA AGG CTG ATG TGA AGG ACA AAG ATG CCG TGG ACG


m214

TGT G


 


Op-T452G-
140
AGA AGG CTG ATG TGA AGG ACG GAG ATG CCG TGG ACG


m216

TGT G


 


Op-T452P-
141
AGA AGG CTG ATG TGA AGG ACC CAG ATG CCG TGG ACG


m217

TGT G


 


op-T452M-
142
AGA AGG CTG ATG TGA AGG ACA TGG ATG CCG TGG ACG


m218

TGT G


 


op-T452F-m219
143
AGA AGG CTG ATG TGA AGG ACT TCG ATG CCG TGG ACG




TGT G


 


op-D461R-6H-
144
CTC TAA TTG CGG CCG CCT ATT AGT GGT GAT GGT GAT


not-m220

GAT GAA AGG CGT CGA TGC ACA CGC CCC TAG CGA TGC




ACA CGT CCA C


 


op-V463Y-6H-
145
CTC TAA TTG CGG CCG CTC ATT AGT GGT GAT GGT GAT


not-m231

GAT GAA AGG CGT CGA TGC AGT AGC CAT CAG CGA TGC




ACA C


 


op-S180T-R-
146
TTC TCT GTG GCC TCG GTC AGG GTC AGC TGC ACG TTC


m232

TGC TGC TG


 


op-D451S-F-
147
AGG AGA AGG CTG ATG TGA AGA GCA CAG ATG CCG TGG


m233

ACG TG


 


op-L313P-
148
ATC GTG TTT ACA GAC CAG GTG CCG AAG TTC CTG TCC


m234-F

CAG GAT GAG


 


op-L313M-
149
ATC GTG TTT ACA GAC CAG GTG ATG AAG TTC CTG TCC


m235-F

CAG GAT GAG


 


op-L179S-
150
TCT GTG GCC TCG GTC AGG CTC GAC TGC ACG TTC TGC


m243-R

TGC TGC AC


 


op-L179I-
151
TCT GTG GCC TCG GTC AGG CTA ATC TGC ACG TTC TGC


m245-R

TGC TGC AC


 


op-L179F-
152
TCT GTG GCC TCG GTC AGG CTA AAC TGC ACG TTC TGC


m246-R

TGC TGC AC


 


FQQ-Mega-
153
ATC GTG ATC TGG GGC TCC TGG GAG TTC ACC AGG ACC


m254

CAG GAG AGC TGC CAG GCC ATC CAG GAG TAC ATG GAC




ACA ACC CTG AAC


 


op-Q268N-
154
ACC CTA GCA TCT ATC TGA ACA CCA ACC AGA GCC CAG


m259-m

TGG CCG CTA C


 


B4-124-R
155
GCC CAG GCA GAA CTC GC


 


op-
156
TCT CGA ACT CCT GCT TAG CCT TAT CTG TGG CCT CGG TCA


L181M, E186D-

TGC TCA GCT GCA CGT TCT GCT GC


m266




 


op-Q268A-
157
ACC CTA GCA TCT ATC TGA ACA CCG CGC AGA GCC CAG


m268-m

TGG CCG CTA C


 


op-344I, 348M-
158
ATC GTG ATC TGG GGC TCC TGG GAG ATC ACC AGG ACC


m271

ATG GAG AGC TGC CAG GCC ATC AAG


 


op-DLSS-m275
159
AGC GGC ATC GTG ATC TGG GGC GAC CTG TCG ATC TCC




TCG ACC ATG GAG AGC TGC CAG GCC


 


op-DLS-m276
160
AGC GGC ATC GTG ATC TGG GGC GAC CTG TCG ATC TCC




AGG ACC ATG GAG AGC TGC CAG


 


Op-K348M-
161
ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC ATG GAG


m279

AGC TGC CAG GCC ATC AAG


 


Op-N344I
162
ATC GTG ATC TGG GGC TCC TGG GAG ATC ACC AGG ACC


K348M-m280

ATG GAG AGC TGC CAG GCC ATC AAG









After finding a PH20 variant with increased enzymatic activity and thermal stability, the His-tag-free cDNA of the PH20 variant was also constructed.
The PH20 variant was constructed as follows using cDNA of the PH20 variant.
The expression of the variant was performed using the EXPICHO™ expression system. When the cell density of EXPICHO™ cells reached 6×106/mL, a plasmid including the wild-type or variant PH20 cDNA inserted in the pcDNA3.4-TOPO vector was transfected into the EXPICHO™ cells using EXPIFECTAMINE™ CHO reagent. As a cell culture medium, EXPICHO™ expression medium (100 to 500 mL) was used. After transfection, the EXPICHO™ cells were cultured with shaking at 130 rpm for a total of 6 days, during which the cells were cultured at 37° C. for 1 day and were further cultured at a lower temperature of 32° C. for 5 days. After completion of the culture, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 min.
The recombinant proteins of the C-terminal His-tag-attached wild-type PH20 and variant PH20, produced in the EXPICHO™ cells, were purified through three-step column chromatography using an AKTA™ prime system (GE Healthcare Systems), and the three-step column chromatography was performed using a HisTrap HP column-Q Sepharose column-phenyl HP column, and a Q Sepharose column-HisTrap HP column-butyl HP column, respectively, depending on the variant.
The purification using the HisTrap HP column, the Q Sepharose column and the phenyl HP column was performed as follows. For protein purification using the HisTrap column, buffer A (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 0.5 M imidazole) were prepared. The protein was bound to the HisTrap column, and the column was flushed with 5 column volumes (CV) of buffer A to remove non-specifically bound proteins. It was confirmed that the conductivity was maintained at a constant level, and then the column was flushed with 5 CV of 20% buffer B to elute the protein. The eluted protein was dialyzed with dialysis buffer (20 mM sodium phosphate, pH 7.5, 50 mM NaCl). For protein purification using the Q Sepharose column, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) were prepared. The protein was bound to the Q Sepharose column, and the column was flushed with 5 CV of buffer A to remove nonspecifically bound proteins, and then was flushed with 5 CV of buffer B at a concentration gradient of 0 to 100% to elute the protein. For protein purification using the phenyl HP column, buffer A (20 mM sodium phosphate, pH 7.0, 1.5 M (NH4)2SO4) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared. The protein was bound to the phenyl column, and the column was flushed with 5 CV of buffer A to remove nonspecifically bound proteins, and then was flushed with 5 CV of buffer B at a concentration gradient of 0 to 100% to elute the protein.
The purification using the Q Sepharose column, the HisTrap HP column and the butyl HP column was performed as follows. For protein purification using the Q Sepharose column, buffer A (20 mM NaPi, 15 mM NaCl, pH 8.0) and buffer B (20 mM NaPi, 500 mM NaCl, pH 8.0) were prepared. To adjust the pH and conductivity of the culture solution to be the same as Buffer A, the pH was titrated to 8 using 1 M Tris buffer, and the conductivity was adjusted to 5 mS/cm or less by adding water (PW) thereto. Then, the culture solution was filtered through a membrane having 0.22-μm pores therein. The protein was bound to the Q Sepharose column, and the column was flushed with 5 CV of buffer A to remove nonspecifically bound proteins and was then flushed with 5 CV of buffer B to elute the target protein. For protein purification using the HisTrap HP column, buffer A (20 mM NaPi, 500 mM NaCl, pH 7.5) and buffer B (20 mM NaPi, 500 mM NaCl, 500 mM Imidazole, pH 7.5) were prepared. The protein sample was bound to the HisTrap HP column, the column was flushed with 10 CV of 7% buffer B to remove non-specifically bound proteins, and the column was then flushed with 3 CV of 40% buffer B to elute the protein. For protein purification using the butyl HP column, buffer A (20 mM NaPi, 1.5 M Ammonium sulfate, pH 7.0) and buffer B (20 mM NaPi, pH 7.0) were prepared. A 3 M ammonium sulfate solution and a protein sample to be loaded onto the column were mixed in a ratio of 1:1 and then the resulting mixture was filtered through a membrane having 0.22-μm pores therein. The protein sample was bound to the butyl HP column, and the column was flushed with 5 CV of buffer A to remove impurities. Then, the target protein was eluted with a linear concentration gradient of 0-100% buffer B, and was dialyzed using a dialysis buffer (20 mM NaPi, 100 mM NaCl, pH 7.0). The variant according to the present invention was purified by the method suggested in the present invention, 10% SDS-PAGE analysis was performed on each purified product, and the results are shown in FIGS. 1 and 3.
The enzymatic activities of wild-type PH20 and variant PH20 were measured by turbidimetric assay.
The turbidimetric assay is a method of measuring the absorbance in the precipitate that is produced when hyaluronic acid is mixed with albumin (BSA). When hyaluronic acid is hydrolyzed by PH20, the absorbance of the precipitate that is produced upon mixing with albumin decreases. The turbidimetric assay is generally performed as follows. Hyaluronidase PH20 (Sigma) was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50 and 60 units/mL and prepared in each tube. The purified protein sample was dissolved in an enzyme diluent buffer (20 mM Tris-HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin), diluted to 100×, 300×, 600×, 1200× and 2400×, and prepared in respective tubes. In fresh tubes, the hyaluronic acid solution having a concentration of 3 mg/mL was diluted 10-fold to a concentration of 0.3 mg/mL so that the volume in each tube became 180 μL. 60 μL of enzyme was added to and mixed with the diluted hyaluronic acid solution and allowed to react at 37° C. for 45 minutes. After completion of the reaction, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.
Methods of measuring the thermal stability of the protein include a method of measuring the aggregation temperature by dynamic light scattering (DLS), a method of measuring the melting temperature (Tm) in real-time PCR using Sypro-Orange dye, and a method of measuring the enzymatic activity after allowing the protein to stand at a predetermined temperature for a predetermined time, etc. In the method of measuring the aggregation temperature by DLS, the aggregation of molecules is measured using light scattering, and thus the sensitivity is high and the aggregation temperature is generally lower than the melting temperature of the protein. Since each variant is prepared as a solution of the same concentration of 0.2 mg/mL and is then measured, the physical properties of each variant can be compared using the resulting value as the aggregation temperature (Philo, J. S. (2009) Cur. Pharm. Biotech. 10, 359-372).
The amino acid sequences of the PH20 variants constructed by substitution or cleavage of the amino acids from the PH20 variant having the sequence of SEQ ID NO: 3 in the present invention are shown in Table 6 below.
In the present invention, the experiment was conducted on a variant in which six histidines were added for protein purification at the C-terminus in the sequence shown in Table 6. It was found that this addition to the C-terminus did not affect the enzyme activity or protein stability. The variant according to the present invention was named as a combination of HM and serial number, and the variants according to Example 3 were named “Hyal2-variant”, “Hyal3-variant”, and “Hyal4-variant”.







TABLE 6







Amino acid sequences of PH20 variants according to present


invention and substitution/cleavage characteristics thereof











SEQ





ID




Name
NO
Substitution
Sequence





Hya12-
163
15 amino acid residues
LNFRAPPVIPNVPFLWAWNAPSEFCLGKF


variant

S341T, W342L, E343S,
DEPLDMSLFSFIGSPRINATGQGVTIFYVD




N344I, R346T, T347S,
RLGYYPYIDSITGVTVNGGIPQKISLQDHL




K348T, S350T, A353Y,
DKAKKDITFYMPVDNLGMAVIDWEEWR




I354L, E356D, M358L,
PTWARNWKPKDVYKNRSIELVQQQNVQ




D359T and T360R,
LSLTEATEKAKQEFEKAGKDFLVETIKLG




T361L are substituted
KLLRPNHLWGYYLFPDCYNHHYKKPGY




from SEQ ID NO: 3,
NGSCFNVEIKRNDDLSWLWNESTALYPSI




cleavage occurs before
YLNTQQSPVAATLYVRNRVREAIRVSKIP




amino acid residue L36
DAKSPLPVFAYTRIVFTDQVLKFLSQDEL




at N-terminus of PH20,
VYTFGETVALGASGIVIWGTLSITTSTET




and cleavage occurs
CQYLKDYLTRLLNPYIINVTLAAKMCSQ




after amino acid residue
VLCQEQGVCIRKNWNSSDYLHLNPDNFA




S490 at C-terminus of
IQLEKGGKFTVRGKPTLEDLEQFSEKFYC




PH20.
SCYSTLSCKEKADVKDTDAVDVCIADGV





CIDAFLKPPMETEEPQIFYNASPSTLS


 


Hya13-
164
17 amino acid residues
LNFRAPPVIPNVPFLWAWNAPSEFCLGKF


variant

S341T, W342L, E343S,
DEPLDMSLFSFIGSPRINATGQGVTIFYVD




N344I, T345S, R346S,
RLGYYPYIDSITGVTVNGGIPQKISLQDHL




T347S, K348E, S350E,
DKAKKDITFYMPVDNLGMAVIDWEEWR




Q352W, A353H,
PTWARNWKPKDVYKNRSIELVQQQNVQ




I354L, K355H, E356D,
LSLTEATEKAKQEFEKAGKDFLVETIKLG




M358L, D359V and
KLLRPNHLWGYYLFPDCYNHHYKKPGY




T360D are substituted
NGSCFNVEIKRNDDLSWLWNESTALYPSI




from SEQ ID NO: 3,
YLNTQQSPVAATLYVRNRVREAIRVSKIP




cleavage occurs before
DAKSPLPVFAYTRIVFTDQVLKFLSQDEL




amino acid residue L36
VYTFGETVALGASGIVIWGTLSISSSEEEC




at N-terminus of PH20,
WHLHDYLVDTLNPYIINVTLAAKMCSQV




and cleavage occurs
LCQEQGVCIRKNWNSSDYLHLNPDNFAI




after amino acid residue
QLEKGGKFTVRGKPTLEDLEQFSEKFYCS




S490 at C-terminus of
CYSTLSCKEKADVKDTDAVDVCIADGVC




PH20.
IDAFLKPPMETEEPQIFYNASPSTLS


 


Hya14-
165
17 amino acid residues
LNFRAPPVIPNVPFLWAWNAPSEFCLGKF


variant

S341T, W342L, E343S,
DEPLDMSLFSFIGSPRINATGQGVTIFYVD




N344I, R346A, T347S,
RLGYYPYIDSITGVTVNGGIPQKISLQDHL




E349A, S350N, Q352T,
DKAKKDITFYMPVDNLGMAVIDWEEWR




A353K, I354V, E356Q,
PTWARNWKPKDVYKNRSIELVQQQNVQ




Y357F, M358V,
LSLTEATEKAKQEFEKAGKDFLVETIKLG




D359S, T360S and
KLLRPNHLWGYYLFPDCYNHHYKKPGY




T361D are substituted
NGSCFNVEIKRNDDLSWLWNESTALYPSI




from SEQ ID NO: 3,
YLNTQQSPVAATLYVRNRVREAIRVSKIP




cleavage occurs before
DAKSPLPVFAYTRIVFTDQVLKFLSQDEL




amino acid residue L36
VYTFGETVALGASGIVIWGTLSITASKAN




at N-terminus of PH20,
CTKVKQFVSSDLNPYIINVTLAAKMCSQ




and cleavage occurs
VLCQEQGVCIRKNWNSSDYLHLNPDNFA




after amino acid residue
IQLEKGGKFTVRGKPTLEDLEQFSEKFYC




S490 at C-terminus of
SCYSTLSCKEKADVKDTDAVDVCIADGV




PH20.
CIDAFLKPPMETEEPQIFYNASPSTLS


 


HM63
166
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




R346M is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTMTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM64
167
One amino acid
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




residue T347Q is
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




substituted from SEQ
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




ID NO: 3, cleavage
AKKDITFYMPVDNLGMAVIDWEEWRPT




occurs before F38
WARNWKPKDVYKNRSIELVQQQNVQLS




amino acid residue at
LTEATEKAKQEFEKAGKDFLVETIKLGKL




N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWENTRQKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM65
168
One amino acid
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




residue K348Q is
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




substituted from SEQ
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




ID NO: 3, cleavage
AKKDITFYMPVDNLGMAVIDWEEWRPT




occurs before F38
WARNWKPKDVYKNRSIELVQQQNVQLS




amino acid residue at
LTEATEKAKQEFEKAGKDFLVETIKLGKL




N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWENTRTQESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM66
169
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S350Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKEQC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM67
170
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K355Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIQEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM69
171
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




M358V is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYVDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM70
172
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L362A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTANPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM71
173
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E343 V is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWVNTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM72
174
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N344F is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWEFTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM73
175
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D359K is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMKTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM74
176
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T360Y is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDYTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM75
177
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T361M is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTMLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM76
178
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q352E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCE





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM77
179
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N363M is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLMPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM78
180
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T84N is substituted
PLDMSLFSFIGSPRINANGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




residue cleavage occurs
AKKDITFYMPVDNLGMAVIDWEEWRPT




before F38 amino acid
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM79
181
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N166K is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKKRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM82
182
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




residue cleavage occurs
AKKDITFYMPVDNLGMAVIDWEEWRPT




before F38 amino acid
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AEKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM83
183
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AQKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM84
184
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354S is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





ASKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM85
185
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354V is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AVKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM86
186
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AAKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM88
187
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354N is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





ANKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM89
188
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354T is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





ATKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM90
189
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E356M is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKMYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM91
190
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E356F is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKFYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM92
191
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E356I is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKIYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM93
192
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E356L is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKLYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM94
193
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E356Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKQYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM95
194
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E356V is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKVYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM96
195
3 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N166K, E343V and
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




T361M are substituted
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




from SEQ ID NO: 3,
AKKDITFYMPVDNLGMAVIDWEEWRPT




cleavage occurs before
WARNWKPKDVYKKRSIELVQQQNVQLS




F38 amino acid residue
LTEATEKAKQEFEKAGKDFLVETIKLGKL




at N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWVNTRTKESCQ





AIKEYMDTMLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM97
196
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




G340Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWQSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM98
197
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S341H is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGHWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM99
198
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




W342I is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSIENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM100
199
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E343Y is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWYNTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM101
200
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T345E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENERTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM102
201
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




R346F is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTFTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM103
202
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T347E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTREKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM104
203
One amino acid
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




residue E349L is
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




substituted from SEQ
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




ID NO: 3, cleavage
AKKDITFYMPVDNLGMAVIDWEEWRPT




occurs before F38
WARNWKPKDVYKNRSIELVQQQNVQLS




amino acid residue at
LTEATEKAKQEFEKAGKDFLVETIKLGKL




N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWENTRTKLSCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM105
204
One amino acid
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




residue S350I is
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




substituted from SEQ
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




ID NO: 3, cleavage
AKKDITFYMPVDNLGMAVIDWEEWRPT




occurs before F38
WARNWKPKDVYKNRSIELVQQQNVQLS




amino acid residue at
LTEATEKAKQEFEKAGKDFLVETIKLGKL




N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWENTRTKEICQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM106
205
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q352G is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCG





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM107
206
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354R is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





ARKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM110
207
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




M358R is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYRDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM111
208
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D359V is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMVTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM112
209
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T360R is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDRTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM114
210
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T345K is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENKRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM115
211
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




R346L is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTLTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM116
212
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T347V is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRVKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM117
213
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E349W is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKWSC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM118
214
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I354W is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AWKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM121
215
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D359Y is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMYTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM125
216
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T347W is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRWKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM126
217
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Y357W is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEWMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM130
218
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




W342D is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSDENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM131
219
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E343Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWQNTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM132
220
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T347H is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRHKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM133
221
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K348F is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTFESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM134
222
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S350D is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKEDC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM135
223
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q352Y is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCY





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM136
224
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




A353E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





EIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM138
225
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




M358Y is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYYDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM139
226
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D359Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMQTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM140
227
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T360L is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDLTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM141
228
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T361E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTELNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM142
229
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N363E is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLEPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM143
230
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




W342H is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSHENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM144
231
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K348D is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTDESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM145
232
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T361H is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTHLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM146
233
No additional
RAPPVIPNVPFLWAWNAPSEFCLGKFDEP




substitution occurs,
LDMSLFSFIGSPRINATGQGVTIFYVDRLG




cleavage occurs before
YYPYIDSITGVTVNGGIPQKISLQDHLDKA




R39 amino acid residue
KKDITFYMPVDNLGMAVIDWEEWRPTW




at N-terminus of PH20,
ARNWKPKDVYKNRSIELVQQQNVQLSLT




and cleavage occurs
EATEKAKQEFEKAGKDFLVETIKLGKLLR




after F468 amino acid
PNHLWGYYLFPDCYNHHYKKPGYNGSC




residue at C-terminus
FNVEIKRNDDLSWLWNESTALYPSIYLNT




of PH20.
QQSPVAATLYVRNRVREAIRVSKIPDAKS





PLPVFAYTRIVFTDQVLKFLSQDELVYTF





GETVALGASGIVIWGSWENTRTKESCQAI





KEYMDTTLNPYIINVTLAAKMCSQVLCQ





EQGVCIRKNWNSSDYLHLNPDNFAIQLEK





GGKFTVRGKPTLEDLEQFSEKFYCSCYST





LSCKEKADVKDTDAVDVCIADGVCIDAF


 


HM147
234
No additional
APPVIPNVPFLWAWNAPSEFCLGKFDEPL




substitution occurs,
DMSLFSFIGSPRINATGQGVTIFYVDRLGY




cleavage occurs before
YPYIDSITGVTVNGGIPQKISLQDHLDKAK




A40 amino acid residue
KDITFYMPVDNLGMAVIDWEEWRPTWA




at N-terminus of PH20,
RNWKPKDVYKNRSIELVQQQNVQLSLTE




and cleavage occurs
ATEKAKQEFEKAGKDFLVETIKLGKLLRP




after F468 amino acid
NHLWGYYLFPDCYNHHYKKPGYNGSCF




residue at C-terminus
NVEIKRNDDLSWLWNESTALYPSIYLNTQ




of PH20.
QSPVAATLYVRNRVREAIRVSKIPDAKSP





LPVFAYTRIVFTDQVLKFLSQDELVYTFG





ETVALGASGIVIWGSWENTRTKESCQAIK





EYMDTTLNPYIINVTLAAKMCSQVLCQE





QGVCIRKNWNSSDYLHLNPDNFAIQLEK





GGKFTVRGKPTLEDLEQFSEKFYCSCYST





LSCKEKADVKDTDAVDVCIADGVCIDAF


 


HM149
235
No additional
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




substitution occurs,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




cleavage occurs before
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




F38 amino acid residue
AKKDITFYMPVDNLGMAVIDWEEWRPT




at N-terminus of PH20,
WARNWKPKDVYKNRSIELVQQQNVQLS




and cleavage occurs
LTEATEKAKQEFEKAGKDFLVETIKLGKL




after D456 amino acid
LRPNHLWGYYLFPDCYNHHYKKPGYNG




residue at C-terminus
SCFNVEIKRNDDLSWLWNESTALYPSIYL




of PH20.
NTQQSPVAATLYVRNRVREAIRVSKIPDA





KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVD


 


HM150
236
2 amino acid residues
RAPPVIPNVPFLWAWNAPSEFCLGKFDEP




S350Q and T360R are
LDMSLFSFIGSPRINATGQGVTIFYVDRLG




substituted from SEQ
YYPYIDSITGVTVNGGIPQKISLQDHLDKA




ID NO: 3, cleavage
KKDITFYMPVDNLGMAVIDWEEWRPTW




occurs before R39
ARNWKPKDVYKNRSIELVQQQNVQLSLT




amino acid residue at
EATEKAKQEFEKAGKDFLVETIKLGKLLR




N-terminus of PH20,
PNHLWGYYLFPDCYNHHYKKPGYNGSC




and cleavage occurs
FNVEIKRNDDLSWLWNESTALYPSIYLNT




after F468 amino acid
QQSPVAATLYVRNRVREAIRVSKIPDAKS




residue at C-terminus
PLPVFAYTRIVFTDQVLKFLSQDELVYTF




of PH20.
GETVALGASGIVIWGSWENTRTKEQCQAI





KEYMDRTLNPYIINVTLAAKMCSQVLCQ





EQGVCIRKNWNSSDYLHLNPDNFAIQLEK





GGKFTVRGKPTLEDLEQFSEKFYCSCYST





LSCKEKADVKDTDAVDVCIADGVCIDAF


 


HM152
237
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFAE




D65A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM153
238
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDA




E66A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM154
239
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




P67A is substituted
ALDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM155
240
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L68A is substituted
PADMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM156
241
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q311A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDAVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM157
242
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




V312A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQALKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM158
243
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L313A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVAKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM159
244
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K314A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLAFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM160
245
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N266A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
ATQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM161
246
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T267A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NAQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM162
247
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q268A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTAQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM163
248
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q269A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQASPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM164
249
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




P271A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSAVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM165
250
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




V272A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPAAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM166
251
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I102A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYADSITGVTVNGGIPQKISLQDHLD




cleavage occurs before
KAKKDITFYMPVDNLGMAVIDWEEWRP




F38 amino acid residue
TWARNWKPKDVYKNRSIELVQQQNVQL




at N-terminus of PH20,
SLTEATEKAKQEFEKAGKDFLVETIKLGK




and cleavage occurs
LLRPNHLWGYYLFPDCYNHHYKKPGYN




after F468 amino acid
GSCFNVEIKRNDDLSWLWNESTALYPSIY




residue at C-terminus
LNTQQSPVAATLYVRNRVREAIRVSKIPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM167
252
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D103A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIASITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM168
253
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S104A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDAITGVTVNGGIPQKISLQDHLD




cleavage occurs before
KAKKDITFYMPVDNLGMAVIDWEEWRP




F38 amino acid residue
TWARNWKPKDVYKNRSIELVQQQNVQL




at N-terminus of PH20,
SLTEATEKAKQEFEKAGKDFLVETIKLGK




and cleavage occurs
LLRPNHLWGYYLFPDCYNHHYKKPGYN




after F468 amino acid
GSCFNVEIKRNDDLSWLWNESTALYPSIY




residue at C-terminus
LNTQQSPVAATLYVRNRVREAIRVSKIPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM169
254
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I105A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSATGVTVNGGIPQKISLQDHLD




cleavage occurs before
KAKKDITFYMPVDNLGMAVIDWEEWRP




F38 amino acid residue
TWARNWKPKDVYKNRSIELVQQQNVQL




at N-terminus of PH20,
SLTEATEKAKQEFEKAGKDFLVETIKLGK




and cleavage occurs
LLRPNHLWGYYLFPDCYNHHYKKPGYN




after F468 amino acid
GSCFNVEIKRNDDLSWLWNESTALYPSIY




residue at C-terminus
LNTQQSPVAATLYVRNRVREAIRVSKIPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM170
255
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDIAFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM171
256
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




F133A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITAYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM172
257
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Y134A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFAMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM173
258
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




V241A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNAEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM174
259
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E242A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVAIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM175
260
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I243 A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEAKRNDDLSWLWNESTALYPSIY




residue at C-terminus
LNTQQSPVAATLYVRNRVREAIRVSKIPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM176
261
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K244A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIARNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM177
262
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L179A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQAS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM178
263
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S180A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLA




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM179
264
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L181A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
ATEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM180
265
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T182A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LAEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM181
266
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T185A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEAAEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM182
267
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




E186A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATAKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM183
268
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K187A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEAAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM184
269
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




K290A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSAIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM185
270
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I291A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKAPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM186
271
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




P292A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIADA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM190
272
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L441A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TASCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM191
273
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S442A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLACKEKADVKDTDAVDVCIADGVCIDA





F


 


HM192
274
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D451A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKATDAVDVCIADGVCIDA





F


 


HM193
275
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDADAVDVCIADGVCIDA





F


 


HM194
276
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D453A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTAAVDVCIADGVCIDA





F


 


HM195
277
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D461A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIAAGVCIDA





F


 


HM196
278
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




G462A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADAVCIDA





F


 


HM197
279
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




V463A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGACIDA





F


 


HM198
280
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N82A is substituted
PLDMSLFSFIGSPRIAATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM199
281
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N166A is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKARSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM203
282
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S104N is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDNITGVTVNGGIPQKISLQDHLD




cleavage occurs before
KAKKDITFYMPVDNLGMAVIDWEEWRP




F38 amino acid residue
TWARNWKPKDVYKNRSIELVQQQNVQL




at N-terminus of PH20,
SLTEATEKAKQEFEKAGKDFLVETIKLGK




and cleavage occurs
LLRPNHLWGYYLFPDCYNHHYKKPGYN




after F468 amino acid
GSCFNVEIKRNDDLSWLWNESTALYPSIY




residue at C-terminus
LNTQQSPVAATLYVRNRVREAIRVSKIPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM204
283
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I105Q is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSQTGVTVNGGIPQKISLQDHLD




cleavage occurs before
KAKKDITFYMPVDNLGMAVIDWEEWRP




F38 amino acid residue
TWARNWKPKDVYKNRSIELVQQQNVQL




at N-terminus of PH20,
SLTEATEKAKQEFEKAGKDFLVETIKLGK




and cleavage occurs
LLRPNHLWGYYLFPDCYNHHYKKPGYN




after F468 amino acid
GSCFNVEIKRNDDLSWLWNESTALYPSIY




residue at C-terminus
LNTQQSPVAATLYVRNRVREAIRVSKIPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM205
284
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q268D is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTDQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM208
285
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




Q268I is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTIQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM210
286
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




I291G is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKGPD




of PH20.
AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM211
287
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




P292D is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIDDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM212
288
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452D is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDDDAVDVCIADGVCIDA





F


 


HM213
289
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452H is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDHDAVDVCIADGVCIDA





F


 


HM214
290
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452K is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDKDAVDVCIADGVCIDA





F


 


HM216
291
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452G is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDGDAVDVCIADGVCIDA





F


 


HM217
292
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452P is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDPDAVDVCIADGVCIDA





F


 


HM218
293
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452M is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDMDAVDVCIADGVCIDA





F


 


HM219
294
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T452F is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDFDAVDVCIADGVCIDA





F


 


HM220
295
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D461R is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIARGVCIDA





F


 


HM231
296
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




V463Y is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGYCIDA





F


 


HM232
297
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S180T is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLT




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM233
298
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




D451S is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKSTDAVDVCIADGVCIDAF


 


HM234
299
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L313P is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVPKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM235
300
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L313M is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQLS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVMKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM243
301
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L179S is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQSS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM245
302
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L179I is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQISL




at N-terminus of PH20,
TEATEKAKQEFEKAGKDFLVETIKLGKLL




and cleavage occurs
RPNHLWGYYLFPDCYNHHYKKPGYNGS




after F468 amino acid
CFNVEIKRNDDLSWLWNESTALYPSIYLN




residue at C-terminus
TQQSPVAATLYVRNRVREAIRVSKIPDAK




of PH20.
SPLPVFAYTRIVFTDQVLKFLSQDELVYTF





GETVALGASGIVIWGSWENTRTKESCQAI





KEYMDTTLNPYIINVTLAAKMCSQVLCQ





EQGVCIRKNWNSSDYLHLNPDNFAIQLEK





GGKFTVRGKPTLEDLEQFSEKFYCSCYST





LSCKEKADVKDTDAVDVCIADGVCIDAF


 


HM246
303
One amino acid residue
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




L179F is substituted
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




from SEQ ID NO: 3,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




cleavage occurs before
AKKDITFYMPVDNLGMAVIDWEEWRPT




F38 amino acid residue
WARNWKPKDVYKNRSIELVQQQNVQFS




at N-terminus of PH20,
LTEATEKAKQEFEKAGKDFLVETIKLGKL




and cleavage occurs
LRPNHLWGYYLFPDCYNHHYKKPGYNG




after F468 amino acid
SCFNVEIKRNDDLSWLWNESTALYPSIYL




residue at C-terminus
NTQQSPVAATLYVRNRVREAIRVSKIPDA




of PH20.
KSPLPVFAYTRIVFTDQVLKFLSQDELVY





TFGETVALGASGIVIWGSWENTRTKESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM254
304
3 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N344F, K348Q and
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




K355Q are substituted
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




from SEQ ID NO: 3,
AKKDITFYMPVDNLGMAVIDWEEWRPT




cleavage occurs before
WARNWKPKDVYKNRSIELVQQQNVQLS




F38 amino acid residue
LTEATEKAKQEFEKAGKDFLVETIKLGKL




at N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWEFTRTQESCQ





AIQEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM261
305
7 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132S, L181A, E186D,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




Q268N, I291L, V312A,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




and T452D are
AKKDISFYMPVDNLGMAVIDWEEWRPT




substituted from SEQ
WARNWKPKDVYKNRSIELVQQQNVQLS




ID NO: 3, cleavage
ATEATDKAKQEFEKAGKDFLVETIKLGK




occurs before F38
LLRPNHLWGYYLFPDCYNHHYKKPGYN




amino acid residue at
GSCFNVEIKRNDDLSWLWNESTALYPSIY




N-terminus of PH20,
LNTNQSPVAATLYVRNRVREAIRVSKLPD




and cleavage occurs
AKSPLPVFAYTRIVFTDQALKFLSQDELV




after F468 amino acid
YTFGETVALGASGIVIWGSWENTRTKESC




residue at C-terminus
QAIKEYMDTTLNPYIINVTLAAKMCSQVL




of PH20.
CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDDDAVDVCIADGVCI





DAF


 


HM262
306
No additional
NFRAPPVIPNVPFLWAWNAPSEFCLGKFD




substitution occurs,
EPLDMSLFSFIGSPRINATGQGVTIFYVDR




cleavage occurs before
LGYYPYIDSITGVTVNGGIPQKISLQDHLD




N37 amino acid residue
KAKKDITFYMPVDNLGMAVIDWEEWRP




at N-terminus of PH20,
TWARNWKPKDVYKNRSIELVQQQNVQL




and cleavage occurs
SLTEATEKAKQEFEKAGKDFLVETIKLGK




after F468 amino acid
LLRPNHLWGYYLFPDCYNHHYKKPGYN




residue at C-terminus
GSCFNVEIKRNDDLSWLWNESTALYPSIY




of PH20.
LNTQQSPVAATLYVRNRVREAIRVSKIPD





AKSPLPVFAYTRIVFTDQVLKFLSQDELV





YTFGETVALGASGIVIWGSWENTRTKESC





QAIKEYMDTTLNPYIINVTLAAKMCSQVL





CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDTDAVDVCIADGVCI





DAF


 


HM263
307
No additional
LNFRAPPVIPNVPFLWAWNAPSEFCLGKF




substitution occurs,
DEPLDMSLFSFIGSPRINATGQGVTIFYVD




cleavage occurs before
RLGYYPYIDSITGVTVNGGIPQKISLQDHL




L36 amino acid residue
DKAKKDITFYMPVDNLGMAVIDWEEWR




at N-terminus of PH20,
PTWARNWKPKDVYKNRSIELVQQQNVQ




and cleavage occurs
LSLTEATEKAKQEFEKAGKDFLVETIKLG




after F468 amino acid
KLLRPNHLWGYYLFPDCYNHHYKKPGY




residue at C-terminus
NGSCFNVEIKRNDDLSWLWNESTALYPSI




of PH20.
YLNTQQSPVAATLYVRNRVREAIRVSKIP





DAKSPLPVFAYTRIVFTDQVLKFLSQDEL





VYTFGETVALGASGIVIWGSWENTRTKES





CQAIKEYMDTTLNPYIINVTLAAKMCSQV





LCQEQGVCIRKNWNSSDYLHLNPDNFAI





QLEKGGKFTVRGKPTLEDLEQFSEKFYCS





CYSTLSCKEKADVKDTDAVDVCIADGVC





IDAF


 


HM266
308
9 amino acid residues
FKAPPVIPNVPFLWAWNAPSEFCLGKFDE




R39K, I105A, T132S,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




L181M, E186D, I291L,
GYYPYIDSATGVTVNGGIPQKISLQDHLD




Q268A, V312A and
KAKKDISFYMPVDNLGMAVIDWEEWRPT




T452D are substituted
WARNWKPKDVYKNRSIELVQQQNVQLS




from SEQ ID NO: 3,
MTEATDKAKQEFEKAGKDFLVETIKLGK




cleavage occurs before
LLRPNHLWGYYLFPDCYNHHYKKPGYN




F38 amino acid residue
GSCFNVEIKRNDDLSWLWNESTALYPSIY




at N-terminus of PH20,
LNTAQSPVAATLYVRNRVREAIRVSKLPD




and cleavage occurs
AKSPLPVFAYTRIVFTDQALKFLSQDELV




after F468 amino acid
YTFGETVALGASGIVIWGSWENTRTKESC




residue at C-terminus
QAIKEYMDTTLNPYIINVTLAAKMCSQVL




of PH20.
CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDDDAVDVCIADGVCI





DAF


 


HM268
309
7 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132A, L181A,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




E186A, Q268A, I291L,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




V312A, and T452D are
AKKDIAFYMPVDNLGMAVIDWEEWRPT




substituted from SEQ
WARNWKPKDVYKNRSIELVQQQNVQLS




ID NO: 3, cleavage
ATEATAKAKQEFEKAGKDFLVETIKLGK




occurs before F38
LLRPNHLWGYYLFPDCYNHHYKKPGYN




amino acid residue at
GSCFNVEIKRNDDLSWLWNESTALYPSIY




N-terminus of PH20,
LNTAQSPVAATLYVRNRVREAIRVSKLPD




and cleavage occurs
AKSPLPVFAYTRIVFTDQALKFLSQDELV




after F468 amino acid
YTFGETVALGASGIVIWGSWENTRTKESC




residue at C-terminus
QAIKEYMDTTLNPYIINVTLAAKMCSQVL




of PH20.
CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDDDAVDVCIADGVCI





DAF


 


HM271
310
2 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




N344I and K348M are
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




substituted from SEQ
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




ID NO: 3, cleavage
AKKDITFYMPVDNLGMAVIDWEEWRPT




occurs before F38
WARNWKPKDVYKNRSIELVQQQNVQLS




amino acid residue at
LTEATEKAKQEFEKAGKDFLVETIKLGKL




N-terminus of PH20,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




and cleavage occurs
SCFNVEIKRNDDLSWLWNESTALYPSIYL




after F468 amino acid
NTQQSPVAATLYVRNRVREAIRVSKIPDA




residue at C-terminus
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




of PH20.
TFGETVALGASGIVIWGSWEITRTMESCQ





AIKEYMDTTLNPYIINVTLAAKMCSQVLC





QEQGVCIRKNWNSSDYLHLNPDNFAIQLE





KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM275
311
10 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S341D, W342L,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




E343S, N344I, T345S,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




R346S, K348M,
AKKDITFYMPVDNLGMAVIDWEEWRPT




K355D, D359E and
WARNWKPKDVYKNRSIELVQQQNVQLS




T361I are substituted
LTEATEKAKQEFEKAGKDFLVETIKLGKL




from SEQ ID NO: 3,
LRPNHLWGYYLFPDCYNHHYKKPGYNG




cleavage occurs before
SCFNVEIKRNDDLSWLWNESTALYPSIYL




F38 amino acid residue
NTQQSPVAATLYVRNRVREAIRVSKIPDA




at N-terminus of PH20,
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




and cleavage occurs
TFGETVALGASGIVIWGDLSISSTMESCQ




after F468 amino acid
AIDEYMETILNPYIINVTLAAKMCSQVLC




residue at C-terminus
QEQGVCIRKNWNSSDYLHLNPDNFAIQLE




of PH20.
KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM276
312
9 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




S341D, W342L,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




E343S, N344I, T345S,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




K348M, K355D,
AKKDITFYMPVDNLGMAVIDWEEWRPT




D359E, and T361I are
WARNWKPKDVYKNRSIELVQQQNVQLS




substituted from SEQ
LTEATEKAKQEFEKAGKDFLVETIKLGKL




ID NO: 3, cleavage
LRPNHLWGYYLFPDCYNHHYKKPGYNG




occurs before F38
SCFNVEIKRNDDLSWLWNESTALYPSIYL




amino acid residue at
NTQQSPVAATLYVRNRVREAIRVSKIPDA




N-terminus of PH20,
KSPLPVFAYTRIVFTDQVLKFLSQDELVY




and cleavage occurs
TFGETVALGASGIVIWGDLSISRTMESCQ




after F468 amino acid
AIDEYMETILNPYIINVTLAAKMCSQVLC




residue at C-terminus
QEQGVCIRKNWNSSDYLHLNPDNFAIQLE




of PH20.
KGGKFTVRGKPTLEDLEQFSEKFYCSCYS





TLSCKEKADVKDTDAVDVCIADGVCIDA





F


 


HM279
313
8 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132S, L181A, E186D,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




Q268N, 1291L, V312A,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




T452D and K348M are
AKKDISFYMPVDNLGMAVIDWEEWRPT




substituted from SEQ
WARNWKPKDVYKNRSIELVQQQNVQLS




ID NO: 3, cleavage
ATEATDKAKQEFEKAGKDFLVETIKLGK




occurs before F38
LLRPNHLWGYYLFPDCYNHHYKKPGYN




amino acid residue at
GSCFNVEIKRNDDLSWLWNESTALYPSIY




N-terminus of PH20,
LNTNQSPVAATLYVRNRVREAIRVSKLPD




and cleavage occurs
AKSPLPVFAYTRIVFTDQALKFLSQDELV




after F468 amino acid
YTFGETVALGASGIVIWGSWENTRTMES




residue at C-terminus
CQAIKEYMDTTLNPYIINVTLAAKMCSQV




of PH20.
LCQEQGVCIRKNWNSSDYLHLNPDNFAI





QLEKGGKFTVRGKPTLEDLEQFSEKFYCS





CYSTLSCKEKADVKDDDAVDVCIADGVC





IDAF


 


HM280
314
9 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132S, L181A, E186D,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




Q268N, I291L, V312A,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




T452D, N344I, and
AKKDISFYMPVDNLGMAVIDWEEWRPT




K348M are substituted
WARNWKPKDVYKNRSIELVQQQNVQLS




from SEQ ID NO: 3,
ATEATDKAKQEFEKAGKDFLVETIKLGK




cleavage occurs before
LLRPNHLWGYYLFPDCYNHHYKKPGYN




F38 amino acid residue
GSCFNVEIKRNDDLSWLWNESTALYPSIY




at N-terminus of PH20,
LNTNQSPVAATLYVRNRVREAIRVSKLPD




and cleavage occurs
AKSPLPVFAYTRIVFTDQALKFLSQDELV




after F468 amino acid
YTFGETVALGASGIVIWGSWEITRTMESC




residue at C-terminus
QAIKEYMDTTLNPYIINVTLAAKMCSQVL




of PH20.
CQEQGVCIRKNWNSSDYLHLNPDNFAIQ





LEKGGKFTVRGKPTLEDLEQFSEKFYCSC





YSTLSCKEKADVKDDDAVDVCIADGVCI





DAF


 


HM287
315
17 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132A, L181A,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




E186A, Q268A, I291L,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




V312A, S341D,
AKKDIAFYMPVDNLGMAVIDWEEWRPT




W342L, E343S, N344I,
WARNWKPKDVYKNRSIELVQQQNVQLS




T345S, R346S,
ATEATAKAKQEFEKAGKDFLVETIKLGK




K348M, K355D,
LLRPNHLWGYYLFPDCYNHHYKKPGYN




D359E, T361I, and
GSCFNVEIKRNDDLSWLWNESTALYPSIY




T452D are substituted
LNTAQSPVAATLYVRNRVREAIRVSKLPD




from SEQ ID NO: 3,
AKSPLPVFAYTRIVFTDQALKFLSQDELV




cleavage occurs before
YTFGETVALGASGIVIWGDLSISSTMESC




F38 amino acid residue
QAIDEYMETILNPYIINVTLAAKMCSQVL




at N-terminus of PH20,
CQEQGVCIRKNWNSSDYLHLNPDNFAIQ




and cleavage occurs
LEKGGKFTVRGKPTLEDLEQFSEKFYCSC




after F468 amino acid
YSTLSCKEKADVKDDDAVDVCIADGVCI




residue at C-terminus
DAF




of PH20.



 


HM288
316
16 amino acid residues
FRAPPVIPNVPFLWAWNAPSEFCLGKFDE




T132A, L181A,
PLDMSLFSFIGSPRINATGQGVTIFYVDRL




E186A, Q268A, I291L,
GYYPYIDSITGVTVNGGIPQKISLQDHLDK




V312A, S341D,
AKKDIAFYMPVDNLGMAVIDWEEWRPT




W342L, E343S, N344I,
WARNWKPKDVYKNRSIELVQQQNVQLS




T345S, K348M,
ATEATAKAKQEFEKAGKDFLVETIKLGK




K355D, D359E, T361I
LLRPNHLWGYYLFPDCYNHHYKKPGYN




and T452D are
GSCFNVEIKRNDDLSWLWNESTALYPSIY




substituted from SEQ
LNTAQSPVAATLYVRNRVREAIRVSKLPD




ID NO: 3, cleavage
AKSPLPVFAYTRIVFTDQALKFLSQDELV




occurs before F38
YTFGETVALGASGIVIWGDLSISRTMESC




amino acid residue at
QAIDEYMETILNPYIINVTLAAKMCSQVL




N-terminus of PH20,
CQEQGVCIRKNWNSSDYLHLNPDNFAIQ




and cleavage occurs
LEKGGKFTVRGKPTLEDLEQFSEKFYCSC




after F468 amino acid
YSTLSCKEKADVKDDDAVDVCIADGVCI




residue at C-terminus
DAF




of PH20.









Example 2. Characterization of PH20 Variants According to Present Invention
Further study on the structure and function of the protein was conducted through research on variants including the cleavage at the N-terminal and C-terminal based on the amino acid sequence of SEQ ID NO: 3. As a result of the expression amount and activity analysis of the prepared variant, the aggregation temperature is shown in Table 7.
Expression level and specific activity were analyzed by the turbidimetric assay described in Example 1. The results of the assay are shown. At this time, activity in the culture solution exceeding 300 unit/mL was marked as “>LOQ”, and activity after purification exceeding 15 unit/μg was marked as “>LOQ” based on the limit of quantification (LOQ) set for each of the activity in the culture solution and the activity after purification. In the opposite case, the inequality sign was changed. The expression level and limits of quantification (LOQ) of the activity analysis, and test results based thereon are shown in Table 7. The aggregation temperature of wild-type PH20 (L36-Y482) of SEQ ID NO: 1 is 46.5° C., and the aggregation temperature of the PH20 variant (F38-F468) of SEQ ID NO: 3 is 51° C.







TABLE 7







Expression level, specific activity, and aggregation 


temperature of PH20 variants according to present invention

















Expression
Specific













Change from
level
activity
Aggrega-



Sequence ID NO. 3
(LOQ:
(LOQ:
tion














Amino Acid
Start
End
300
15
point 


Variants
substitution
from
with
units/mL)
units/μg)
(° C.)





HM63
R346M
F38
F468
>LOQ
>LOQ
52° C.


HM64
T347Q
F38
F468
>LOQ
>LOQ
50° C.


HM65
K348Q
F38
F468
>LOQ
>LOQ
51° C.


HM66
S350Q
F38
F468
>LOQ
>LOQ
56° C.


HM67
K355Q
F38
F468
>LOQ
>LOQ
50° C.


HM69
M358V
F38
F468
>LOQ
>LOQ
50° C.


HM70
L362A
F38
F468
>LOQ
>LOQ
48° C.


HM71
E343V
F38
F468
>LOQ
>LOQ
50° C.


HM72
N344F
F38
F468
>LOQ
>LOQ
52° C.


HM73
D359K
F38
F468
>LOQ
>LOQ
50° C.


HM74
T360Y
F38
F468
>LOQ
>LOQ
50° C.


HM75
T361M
F38
F468
>LOQ
>LOQ
49° C.


HM76
Q352E
F38
F468
>LOQ
>LOQ
52° C.


HM77
N363M
F38
F468
>LOQ
>LOQ
58° C.


HM78
T84N
F38
F468
>LOQ
>LOQ
48° C.


HM79
N166K
F38
F468
>LOQ
>LOQ
49° C.


HM82
I354E
F38
F468
>LOQ
>LOQ
49° C.


HM83
I354Q
F38
F468
>LOQ
>LOQ
49° C.


HM84
I354S
F38
F468
>LOQ
>LOQ
48° C.


HM85
I354V
F38
F468
>LOQ
>LOQ
51° C.


HM86
I354A
F38
F468
>LOQ
>LOQ
49° C.


HM88
I354N
F38
F468
>LOQ
>LOQ
49° C.


HM89
I354T
F38
F468
>LOQ
>LOQ
49° C.


HM90
E356M
F38
F468
>LOQ
>LOQ
50° C.


HM91
E356F
F38
F468
>LOQ
>LOQ
49° C.


HM92
E356I
F38
F468
>LOQ
>LOQ
49° C.


HM93
E356L
F38
F468
>LOQ
>LOQ
49° C.


HM94
E356Q
F38
F468
>LOQ
>LOQ
50° C.


HM95
E356V
F38
F468
>LOQ
>LOQ
48° C.


HM96
N166K, E343V,
F38
F468
>LOQ
>LOQ
48° C.



T361M







HM97
G340Q
F38
F468
>LOQ
>LOQ
50° C.


HM98
S341H
F38
F468
>LOQ
>LOQ
51° C.


HM99
W342I
F38
F468
>LOQ
>LOQ
51° C.


HM100
E343Y
F38
F468
>LOQ
>LOQ
50° C.


HM101
T345E
F38
F468
>LOQ
>LOQ
51° C.


HM102
R346F
F38
F468
>LOQ
>LOQ
51° C.


HM103
T347E
F38
F468
>LOQ
>LOQ
50° C.


HM104
E349L
F38
F468
>LOQ
>LOQ
50° C.


HM105
S350I
F38
F468
>LOQ
>LOQ
50° C.


HM106
Q352G
F38
F468
>LOQ
>LOQ
49° C.


HM107
I354R
F38
F468
>LOQ
>LOQ
48° C.


HM110
M358R
F38
F468
>LOQ
>LOQ
48° C.


HM111
D359V
F38
F468
>LOQ
>LOQ
50° C.


HM112
T360R
F38
F468
>LOQ
>LOQ
52° C.


HM114
T345K
F38
F468
>LOQ
>LOQ
50° C.


HM115
R346L
F38
F468
>LOQ
>LOQ
52° C.


HM116
T347V
F38
F468
>LOQ
>LOQ
49° C.


HM117
E349W
F38
F468
>LOQ
>LOQ
49° C.


HM118
I354W
F38
F468
>LOQ
>LOQ
51° C.


HM121
D359Y
F38
F468
>LOQ
>LOQ
49° C.


HM125
T347W
F38
F468
>LOQ
>LOQ
51° C.


HM126
Y357W
F38
F468
>LOQ
>LOQ
55° C.


HM130
W342D
F38
F468
>LOQ
>LOQ
51° C.


HM131
E343Q
F38
F468
>LOQ
>LOQ
50° C.


HM132
T347H
F38
F468
>LOQ
>LOQ
51° C.


HM133
K348F
F38
F468
>LOQ
>LOQ
49° C.


HM134
S350D
F38
F468
>LOQ
>LOQ
54° C.


HM135
Q352Y
F38
F468
>LOQ
>LOQ
51° C.


HM136
A353E
F38
F468
>LOQ
>LOQ
49° C.


HM138
M358Y
F38
F468
>LOQ
>LOQ
48° C.


HM139
D359Q
F38
F468
>LOQ
>LOQ
50° C.


HM140
T360L
F38
F468
>LOQ
>LOQ
49° C.


HM141
T361E
F38
F468
>LOQ
>LOQ
48° C.


HM142
N363E
F38
F468
>LOQ
>LOQ
50° C.


HM143
W342H
F38
F468
>LOQ
>LOQ
50° C.


HM144
K348D
F38
F468
>LOQ
>LOQ
50° C.


HM145
T361H
F38
F468
>LOQ
>LOQ
49° C.


HM146
—
R39
F468
>LOQ
>LOQ
52° C.


HM147
—
A40
F468
>LOQ
>LOQ
53° C.


HM149
—
F38
D456
>LOQ
>LOQ
54° C.


HM150
S350Q, T360R
R39
F468
>LOQ
>LOQ
50° C.


HM152
D65A
F38
F468
>LOQ
>LOQ
51° C


HM153
E66A
F38
F468
>LOQ
>LOQ
51° C.


HM154
P67A
F38
F468
>LOQ
>LOQ
50° C.


HM155
L68A
F38
F468
>LOQ
>LOQ
51° C.


HM156
Q311A
F38
F 468
>LOQ
>LOQ
50° C.


HM157
V312A
F38
F468
>LOQ
>LOQ
56° C.


HM158
L313A
F38
F468
>LOQ
>LOQ
55° C.


HM159
K314A
F38
F468
>LOQ
>LOQ
49° C.


HM160
N266A
F38
F468
>LOQ
>LOQ
49° C.


HM161
T267A
F38
F468
>LOQ
>LOQ
50° C.


HM162
Q268A
F38
F468
>LOQ
>LOQ
51° C.


HM163
Q269A
F38
F468
>LOQ
>LOQ
51° C.


HM164
P271A
F38
F468
>LOQ
>LOQ
51° C.


HM165
V272A
F38
F468
>LOQ
>LOQ
52° C.


HM166
I102A
F38
F468
>LOQ
>LOQ
49° C.


HM167
D103A
F38
F468
>LOQ
>LOQ
53° C.


HM168
S104A
F38
F468
>LOQ
>LOQ
51° C.


HM169
I105A
F38
F468
>LOQ
>LOQ
51° C.


HM170
T132A
F38
F468
>LOQ
>LOQ
51° C.


HM171
F133A
F38
F468
>LOQ
>LOQ
50° C.


HM172
Y134A
F38
F468
>LOQ
>LOQ
52° C.


HM173
V241A
F38
F468
>LOQ
>LOQ
50° C.


HM174
E242A
F38
F468
<LOQ
>LOQ
55° C.


HM175
I243A
F38
F468
>LOQ
>LOQ
49° C.


HM176
K244A
F38
F468
>LOQ
>LOQ
50° C.


HM177
L179A
F38
F468
>LOQ
>LOQ
54° C.


HM178
S180A
F38
F468
>LOQ
>LOQ
50° C.


HM179
L181A
F38
F468
>LOQ
>LOQ
50° C.


HM180
T182A
F38
F468
>LOQ
>LOQ
50° C.


HM181
T185A
F38
F468
>LOQ
>LOQ
50° C.


HM182
E186A
F38
F468
>LOQ
>LOQ
51° C.


HM183
K187A
F38
F468
>LOQ
>LOQ
50° C.


HM184
K290A
F38
F468
>LOQ
>LOQ
50° C.


HM185
I291A
F38
F468
>LOQ
>LOQ
54° C.


HM186
P292A
F38
F468
>LOQ
>LOQ
52° C.


HM190
L441A
F38
F468
>LOQ
>LOQ
50° C.


HM191
S442A
F38
F468
>LOQ
>LOQ
54° C.


HM192
D451A
F38
F468
>LOQ
>LOQ
54° C.


HM193
T452A
F38
F468
>LOQ
>LOQ
53° C.


HM194
D453A
F38
F468
>LOQ
>LOQ
49° C.


HM195
D461A
F38
F468
>LOQ
>LOQ
49° C.


HM196
G462A
F38
F468
>LOQ
>LOQ
50° C.


HM197
V463A
F38
F468
>LOQ
>LOQ
49° C.


HM198
N82A
F38
F468
>LOQ
>LOQ
49° C.


HM199
N166A
F38
F468
>LOQ
>LOQ
50° C.


HM203
S104N
F38
F468
>LOQ
>LOQ
48° C.


HM204
I105Q
F38
F468
>LOQ
>LOQ
51° C.


HM205
Q268D
F38
F468
>LOQ
>LOQ
55° C.


HM208
Q268I
F38
F468
<LOQ
>LOQ
52° C.


HM210
I291G
F38
F468
<LOQ
>LOQ
55° C.


HM211
P292D
F38
F468
<LOQ
>LOQ
53° C.


HM212
T452D
F38
F468
>LOQ
>LOQ
50° C.


HM213
T452H
F38
F468
>LOQ
>LOQ
50° C.


HM214
T452K
F38
F468
>LOQ
>LOQ
52° C.


HM216
T452G
F38
F468
>LOQ
>LOQ
54° C.


HM217
T452P
F38
F468
>LOQ
>LOQ
52° C.


HM218
T452M
F38
F468
>LOQ
>LOQ
52° C.


HM219
T452F
F38
F468
>LOQ
>LOQ
53° C.


HM220
D461R
F38
F468
>LOQ
>LOQ
48° C.


HM231
V463Y
F38
F468
>LOQ
>LOQ
51° C.


HM232
S180T
F38
F468
>LOQ
>LOQ
51° C.


HM233
D451S
F38
F468
>LOQ
>LOQ
51° C.


HM234
L313P
F38
F468
>LOQ
>LOQ
49° C.


HM235
L313M
F38
F468
>LOQ
>LOQ
52° C.


HM243
L179S
F38
F468
>LOQ
>LOQ
52° C.


HM245
L179I
F38
F468
>LOQ
>LOQ
48° C.


HM246
L179F
F38
F468
>LOQ
>LOQ
55° C.


HM254
N344F, K348Q,
F38
F468
>LOQ
>LOQ
53° C.



K355Q







HM261
T132S, L181A,
F38
F468
>LOQ
>LOQ
56° C.



E186D, Q268N,








I291L, V312A,








T452D







HM262
—
N37
F468
>LOQ
>LOQ
50° C.


HM263
—
L36
F468
>LOQ
>LOQ
49° C.


HM266
R39K, I105A,
F38
F468
>LOQ
>LOQ
57° C.



T132S, L181M,








E186D, Q268A,








I291L, V312A,








T452D







HM268
T132A, L181A,
F38
F468
>LOQ
>LOQ
53° C.



E186A, Q268A,








I291L, V312A,








T452D







HM271
N344I, K348M
F38
F468
>LOQ
>LOQ
51° C.


HM275
S341D, W342L,
F38
F468
>LOQ
>LOQ
48° C.



E343S, N344I,








T345S, R346S,








K348M, K355D,








D359E, T361I







HM276
S341D, W342L,
F38
F468
>LOQ
>LOQ
48° C.



E343S, N344I,








T345S, K348M,








K355D, D359E,








T361I







HM279
T132S, L181A,
F38
F468
>LOQ
>LOQ
56° C.



E186D, Q268N,








I291L, V312A,








K348M, T452D







HM280
T132S, L181A,
F38
F468
<LOQ
>LOQ
59° C.



E186D, Q268N,








I291L, V312A,








N344I, K348M,








T452D







HM287
T132A, L181A,
F38
F468
<LOQ
>LOQ
48° C.



E186A, Q268A,








I291L, V312A,








S341D, W342L,








E343S, N344I,








T345S, R346S,








K348M, K355D,








D359E, T361I,








T452D







HM288
T132A, L181A,
F38
F468
<LOQ
>LOQ
48° C.



E186A, Q268A,








I291L, V312A,








S341D, W342L,








E343S, N344I,








T345S, K348M,








K355D, D359E,








T361I, T452D









As can be seen from Table 7 above, among the variants having the amino acid sequence of SEQ ID NO: 3, a total of 133 types of variants having one amino acid residue substitution, namely, HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM97, HM98, HM99, HM100, HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144, HM145, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245 and HM246, were variants that still maintain the activity in the purified fraction obtained after purification and have an aggregation temperature of 48 to 58° C. and thus exhibit excellent thermal stability. Thereamong, a total of 65 types of variants, namely HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM98, HM99, HM100, HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144 and HM145, are variants that are mutated at one of substitution sites in the sequence of SEQ ID NO: 3 from PH20, and have an aggregation temperature of 48° C. to 58° C. There among, a total of 68 types of variants, namely HM97, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245 and HM246, are variants that are mutated at one position of sites other than the substitution sites in SEQ ID NO: 3 from PH20, and have an aggregation temperature of 48° C. to 56° C.
As a result, it can be seen that the variant having substitution at one position from SEQ ID NO: 3 has a higher aggregation temperature than that of wild-type PH20 (L36-Y482) of SEQ ID NO: 1, regardless of the substitution position. However, among them, HM174, HM208, HM210 and HM211 were found to have lower activity in the culture solution than 300 unit/mL, which is LOQ, but have higher activity after purification than 15 unit/μg, which is LOQ. In this case, it is considered that the characteristics of the variant itself cannot be accurately analyzed when the activity of the variant is measured only in the culture solution.
In addition, as shown in Table 7 above, among the variants having the amino acid sequence of SEQ ID NO: 3, HM146, HM147, HM149, HM262 and HM263 retain the same mutations as the variant having the amino acid sequence of SEQ ID NO: 3, that is, substitution of amino acid residues, but further include cleavage at the N-terminus and the C-terminus, which means that the expression and activity of proteins in the variants having the amino acid sequence of SEQ ID NO: 3 are not affected by further cleavage at the N-terminus and the C-terminus. These variants have an aggregation temperature of 49° C. to 53° C., which is not very different from that of the variant of SEQ ID NO: 3, which means that the physical properties of the variants were also unaffected by further cleavage at the N-terminus and the C-terminus.
In addition, in the variants having the amino acid sequence of SEQ ID NO: 3, a total of 13 types of variants, namely HM96, HM150, HM254, HM261, HM266, HM268, HM271, HM275, HM276, HM279, HM280, HM287 and HM288, which are variants including one or more amino acid substitutions and cleavages among those listed in Table 7 above, successfully expressed proteins, further retained enzyme activity, and had an aggregation temperature of 48° C. to 59° C. This means that even in the case of such multiple substitutions, the activity and physical properties of proteins were maintained. However, multiple substitutions exhibited unpredictable enzymatic activity and aggregation temperature which are unpredictable only with the combination of characteristics that are obtained in each single substitution constituting the same.
Example 3. Analysis of Activity of Variants Substituted with Sequences of Hyal2, Hyal3, and Hyal4
The amino acid sequences of Hyal2 (TTSTETCQYLKDYLTRL) (SEQ ID NO: 317), Hyal3 (SSSEEECWHLHDYLVDT) (SEQ ID NO: 318), and Hyal4 (TASKANCTKVKQFVSSD) (SEQ ID NO: 319), which are the corresponding parts of hyaluronidases present in humans, other than Hyal1, in place of M345 to 1361 sites in the amino acid sequence of the wild-type PH20 of SEQ ID NO: 1, were introduced into the M345 to 1361 sites to investigate how the stability of proteins changes.
The variants constructed by substituting the M345 to 1361 sites of mature wild-type PH20 (L36-S490) with corresponding sequences of Hyal2, Hyal3, and Hyal4, are referred to as “Hyal2-variant”, “Hyal3-variant” and “Hyal4-variant”, respectively.
The Hyal2-variant, Hyal3-variant and Hyal4-variant were constructed, and then the thermal stability of these variants was analyzed (see FIG. 2). As a result, the aggregation temperature of the Hyal3-variant measured by DLS was 48° C., which was 1.5° C. higher than 46.5° C., which is the aggregation temperature of the wild-type PH20, which means that the thermal stability was increased.
In addition, in order to confirm whether or not these variants were expressed in EXPICHO™ cell culture, the variants were purified by the same method using a HisTrap column, and the expression levels of proteins were compared by SDS-PAGE analysis. The result showed that the expression level of the Hyal3-variant was the highest, followed by the Hyal2-variant and Hyal4-variant in descending order.
Example 4. Analysis of Thermal Stability of Variants According to Present Invention
SDS-PAGE analysis was performed to confirm the thermal stability of the variants according to the present invention. The purified wild-type PH20 of SEQ ID NO: 1 (L36-Y482) and the purified protein of SEQ ID NO: 3 (F38-F468) of the PH20 variants according to the present invention were stored at 42° C. for 7 days, followed by 10% SDS-PAGE analysis under reducing and non-reducing conditions (FIG. 4).
As a result, wild-type PH20 (L36-Y482) was observed to aggregate (Lane G in FIG. 4), whereas variant PH20 (F38-F468) of SEQ ID NO: 3 did not aggregate (Lane H in FIG. 4). This difference in aggregation was found to be due to the difference in aggregation temperature between the two proteins. Accordingly, the variant according to the present invention was considered to exhibit higher thermal stability and thus is expected to be widely industrially applicable due to the high aggregation temperature thereof compared to the wild-type PH20.
Example 5. Enzyme Kinetics Analysis of Variants According to Present Invention
In order to analyze the enzyme kinetics of the variants according to the present invention, the enzyme activity was measured by the Morgan-Elson method (Takahashi, T. et al (2003) Anal. Biochem. 322:257-263). The Morgan-Elson method is a colorimetric method that assays red substances (at 545 nm) produced by the reaction of the reducing end of N-acetyl-D-glucosamine (GlcNAc) produced upon hydrolysis of hyaluronic acid by hyaluronidase with para-dimethylaminobenzaldehyde (DMAB), which is an Ehrlich's Reagent. N-acetyl-D-glucosamine (GlcNAc, Sigma) diluted to 0.25, 0.50, 0.75, 1.00 or 1.25 mM in dilution buffer solution (0.1 M NaPi, 0.1 M NaCl, 1.5 mM saccharic acid 1,4-lactone, pH 5.35) was reduced by treatment with tetraborate in each test tube, and then DMAB was added to induce colorimetric reaction. After the reaction, absorbance was measured at 545 nm to create a standard reaction curve for GlcNAc. Hyaluronic acid as a substrate was diluted to 0.54, 0.65, 0.87, 1.23 or 2.17 μM in a dilution buffer solution in each test tube, and hyaluronidase was added thereto, followed by reaction at 37° C. for 5 minutes and heating at 100° C. for 5 minutes to terminate the enzyme reaction. The resultant sample after enzyme reaction was reduced by treatment with tetraborate, and DMAB was added to induce colorimetric reaction. After the reaction, absorbance was measured at 545 nm, and enzyme activity was measured using the standard reaction curve of GlcNAc above. The enzyme kinetics of the wild-type PH20 of SEQ ID NO: 1 and the PH20 variant according to the present invention were analyzed using this method. As a result, the linearity of the Lineweaver-Burk curve was detected, which means that the PH20 variant according to the present invention follows the Michaelis-Menten enzyme kinetics equation.
Table 8 shows Vmax (maximum enzyme reaction rate), KM(50% substrate concentration), kcat (substrate conversion rate), and kcat/KM (enzyme catalyst efficiency) obtained as the result of analysis of enzyme kinetics regarding wild-type PH20 (L36-Y482) of SEQ ID NO: 1, variant PH20 (F38-F468) of SEQ ID NO: 3, HM261, and HM268. It can be seen that, as the value of KM decreases, the substrate-binding capacity of the enzyme increases, and as the value of kcat increases, the substrate conversion rate of the enzyme increases, so kcat/KM (enzyme catalyst efficiency) of each PH20 variant is higher than that of the wild-type PH20. In addition, the kcat of each of SEQ ID NO: 3, HM261 and HM268 is greater than that of the wild-type PH20 of SEQ ID NO: 1, and thus the substrate conversion rate of the enzyme is larger than that of the wild-type PH20 of SEQ ID NO: 1, so the industrial availability of each PH20 variant is greater than that of the wild-type PH20.







TABLE 8







Results of enzyme kinetics analysis of PH20


variants according to present invention












Vmax
KM
kcat




(μM/sec)
(μM)
(1/sec)
kcat/ KM





PH20
4.5 ± 0.5
2.0 ± 0.3
30.6 ± 3.0
15.1 ± 1.1


SEQ ID NO: 1






(L36-Y482)






SEQ ID NO: 3
3.7 ± 0.3
1.3 ± 0.0
47.6 ± 3.7
36.8 ± 2.0


(F38-F468)






HM261
5.2 ± 0.5
1.4 ± 0.2
33.9 ± 3.0
23.7 ± 2.2


HM268
2.9 ± 0.5
0.9 ± 0.2
37.1 ± 6.4
40.0 ± 3.4