Claims for Patent: 5,891,671

Summary for Patent: 5,891,671

Title:	Method for cleaving chimeric protein using processing enzyme
Abstract:	A chimeric protein is provided which comprises a target protein and has been designed so as to be easily cleaved by a processing enzyme, permitting a target protein to be efficiently recovered. The chimeric protein is represented by the following formula: wherein A represents a protective peptide; B represents a target peptide; and L represents a linker peptide having the sequence X.sub.1 -X.sub.2 -(Pro, Lys, or Arg)-Arg, wherein X.sub.1 and X.sub.2 represent any amino acid, in its C-terminal region and a domain rich in His in its N-terminal region.
Inventor(s):	Suzuki; Yuji (Ashikaga, JP), Magota; Koji (Takatsuki, JP), Masuda; Toyofumi (Ibaraki, JP)
Assignee:	Suntory Limited (Osaka, JP)
Application Number:	08/811,028
Patent Claims:	1. A chimeric protein of the formula: wherein A is a protective peptide; B is a target peptide; and L is a linker peptide having the sequence X.sub.1 -X.sub.2 -(Pro, Lys, or Arg)-Arg in its C-terminal region and a domain rich in his in its N-terminal region, wherein X.sub.1 is any amino acid except Asp and Glu, and X.sub.2 is any amino acid except Pro. 2. The chimeric protein according to claim 1, wherein the domain rich in His in the N-terminal region has the sequence: [(His).sub.4 -Pro-Gly SEQ ID NO: 49].sub.n wherein n is 1 to 6. 3. The chimeric protein according to claim 1, wherein any 1 to 5 amino acids exist between the amino acid sequence in the N-terminal region and the amino acid sequence in the C-terminal region. 4. The chimeric protein according to claim 1, wherein, in the C-terminal region, X.sub.1 is Arg, Lys or His and X.sub.2 is His or Phe. 5. The chimeric protein according to claim 1, wherein A represents a polypeptide not containing the amino acid sequence: Pro-Arg, Arg-Arg, and Lys-Arg. 6. The chimeric protein according to claim 1, wherein when A represents a polypeptide containing the amino acid sequence: Pro-Arg, Arg-Arg, or Lys-Arg, at least one amino acid in said amino acid sequence has been substituted with a different amino acid so as for A not to contain said amino acid sequence. 7. The chimeric protein according to claim 1, wherein A represents a polypeptide of not more than 220 amino acid residues. 8. The chimeric protein according to claim 7, wherein A represents a peptide of amino acids 1-97, 1-117, or 1-139 on the N-terminal side of .beta.-galactosidase derived from E. coli. 9. The chimeric protein according to claim 1, wherein any cysteine in A has been substituted with a different amino acid. 10. The chimeric protein according to claim 9, wherein the different amino acid is serine. 11. The chimeric protein according to claim 1, wherein B represents a human parathyroid hormone or a derivative having the biological activity of human parathyroid hormone. 12. The chimeric protein according to claim 11, wherein the human parathyroid hormone derivative comprises a fragment comprising amino acids from 1 or 3 to 31, 34, 37, 38, or 84 in the N-terminal of a naturally occurring human parathyroid hormone, or a fragment comprising said amino acid residues with Gly being further added to the C-terminal. 13. The chimeric protein according to claim 1, wherein B represents glucagon-like-peptide 1 or a derivative having the biological activity of glucagon-like-peptide 1. 14. A process for producing the chimeric protein according to claim 1, comprising the steps of: transforming an expression vector containing DNA encoding the chimeric protein into a host cell; culturing the resultant transformants; and harvesting the chimeric protein from the culture. 15. A process for producing a target peptide (B), wherein a processing enzyme acts on the chimeric protein according to claim 1 and cleaves a peptide bond between the C-terminal of the linker peptide (L) and the N-terminal of the target peptide (B) to produce the target peptide (B). 16. The process according to claim 15, wherein the processing enzyme is a member selected from Kex2 protease, furin, a prohormone convertase 1/3 (PC1/3), a prohormone convertase 2 (PC2), and derivatives having the enzymatic activity of such processing enzymes. 17. The process according to claim 15, wherein the target peptide (B) is recovered by isoelectric precipitation.

Details for Patent 5,891,671

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2016-03-04