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AssigneeThe Scripps Research Institute
InventorsJohn H. Griffin, Andrew J. Gale, Elizabeth D. Getzoff, Jean-Luc Pellequer
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The present invention relates to methods of introducing one or more cysteine residues into a polypeptide which permit the stabilization of the polypeptide by formation of at least one bond, preferably a disulfide bond, between different domains of the polypeptide. The invention also relates to polypeptides containing such introduced cysteine residue(s), nucleic acids encoding such polypeptides and pharmaceutical compositions comprising such polypeptides or nucleic acids. The invention also relates to vectors, viral particles and host cells containing such nucleic acids, and methods of using them to produce the polypeptides of the invention. Exemplified polypeptides include plasma proteins, including hepatocyte growth factor activator and plasma hyaluronin binding protein, as well as blood coagulation factors, such as Factor VIII, Factor V, Factor XII and prothrombin.
A method of stabilizing a polypeptide which is the product of a single gene in nature by introducing one or more cysteines comprising the steps of:
A method of stabilizing a polypeptide which is the product of a single gene in nature by introducing one or more cysteines comprising the steps of:
A polypeptide which is the product of a single gene in nature which has been mutated by introducing at least one cysteine, wherein the introduction of said cysteine permits the formation of at least one intramolecular, interdomain disulfide bridge with another cysteine, which increases the stability of the mutant polypeptide as compared to that of the polypeptide which does not contain said introduced cysteine.
A mutant Factor VIII which comprises at least one pair of cysteines located at residues selected from the group consisting of Met 662-Asp 1828, Ser 268-Phe 673, Ile 312-Pro 672, Ser 313-Ala 644, Met 662-Lys 1827, Tyr 664-Thr 1826, Pro 264-Gln 645, Arg 282-Thr 522, Ser 285-Phe 673, His 311-Phe 673, Ser 314-Ala 644, Ser 314-Gln 645, Val 663-Glu 1829, Asn 694-Pro 1980, and Ser 695-Glu 1844.
A composition comprising a mutant Factor VIII of claim 12, 13 or 19.
A pharmaceutical composition comprising a mutant Factor VIII of claim 12, 13 or 19 and a pharmaceutically acceptable carrier.
A nucleic acid coding for a mutant Factor VIII according to claim 12, 13 or 19.
A mutant Factor VIII comprising:
A factor V mutant which comprises at least one pair of cysteines located at residues selected from the group consisting of His609-Glu1691, Leu238-Gln590, His253-Asp469, Ala257-Met618, and Leu283-Met618.
A mutant prothrombin comprising one or more introduced cysteines wherein at least one of the introduced cysteines is capable of forming a disulfide bond with another cysteine residue, wherein said disulfide bond is formed between the Kringle 2 and protease domains of said prothrombin mutant.
A mutant prothrombin which comprises at least one pair of cysteines located at residues selected from the group consisting of Asp 261-Arg 443, Asp 261-Lys 567, and His 205-Lys 572.
Dependencies are inferred from the claim text.