Claims for Patent: 4,543,329

Summary for Patent: 4,543,329

Title:	Process for the specific cleavage of protein sequences from proteins
Abstract:	A process for cleavage of a protein (a) sequence from a protein containing the tetrapeptide sequence Pro-Xyz-Gly-Pro, wherein Xyz can be any desired amino acid, and reacting the protein with a collagenase whereby the Xyz-Gly bond is selectively cleaved, thereby producing a protein with the sequence Gly-Pro at its N-terminal end; or (b) containing the sequence Met-Pro; and subsequently selectively cleaving the glycine or methionine residue from the N-terminal end of the product protein with an aminoacylproline aminopeptidase and selectively cleaving the proline residue therefrom with a proline aminopeptidase; or subsequently selectively cleaving the Gly-Pro or Met-Pro residue from the N-terminal end of the produced protein using a postproline dipeptidylaminopeptidase. In a special embodiment, the protein which is split contains the sequence -Zxy-Uvw-Pro- on the carboxy end of the sequence Pro-Xyz-Gly-Pro, wherein Zxy and Uvw are independently any natural amino acid in the genetic code except Pro, whereby after the collagenase reaction, there is produced a protein with the sequence Gly-Pro-Zxy-Uvw-Pro at the N-terminal end. This is split by subsequently selectively cleaving the Pro-Zxy-bond by treating the protein with postproline dipeptidylaminopeptidase (PPDA), thereby producing protein having the sequence Zxy-Uvw-Pro at its N-terminal end, followed by cleaving the Zxy acid from the resultant protein by treating the latter with leucine aminopeptidase.
Inventor(s):	Daum; Joachim (Berlin, DE), Siewert; Gerhard (Berlin, DE), Topert; Michael (Berlin, DE), Seliger; Hartmut (Ulm-Lehr, DE)
Assignee:	Schering Aktiengesellschaft (Berlin and Bergkamen, DE)
Application Number:	06/395,433
Patent Claims:	1. In a process for selectively cleaving a fusion protein produced from a genetically engineered microorganism containing a foreign gene, the fusion protein comprising native protein segment and foreign protein and the cleavage selectively occurring between the native and foreign protein the improvement comprising incorporating the tetrapeptide sequence Pro-Xyz-Gly-Pro between the native and foreign protein, wherein Xyz can be any natural amino acid and its carboxy end is attached to the amino end of Gly, and reacting the fusion protein with a collagenase whereby said Xyz-Gly bond is selectively cleaved, thereby producing a foreign protein with the sequence Gly-Pro at its N-terminal end. 2. The process of claim 1 wherein the collagenase is E.C. 3.4.24.3. (Clostridiopeptidase A). 3. The process of claim 1 further comprising subsequently selectively cleaving said glycine residue from the N-terminal end of the produced protein with an aminoacylproline aminopeptidase. 4. The process of claim 3 wherein the collagenase is E.C. 3.4.24.3, and the aminoacylproline aminopeptidase is aminopeptidase P, E.C. 3.4.11.9. 5. The process of claim 3 further comprising selectively cleaving said terminal Pro therefrom with a proline aminopeptidase. 6. The process of claim 5 wherein the proline aminopeptidase is E.C. 3.4.11.5. 7. The process of claim 5 wherein the cleavage of the glycine residue with an aminoacylproline aminopeptidase and of the proline residue with proline aminopeptidase is conducted in one step. 8. The process of claim 1 further comprising selectively cleaving said Gly-Pro residue from the N-terminal end of the produced foreign protein using postproline dipeptidylaminopeptidase. 9. The process of claim 1 wherein the foreign protein itself is terminated by the sequence Uvw-Pro at its N-terminal end, and which further comprises incorporating the amino acid Zxy on the carboxy end of the sequence Pro-Xyz-Gly-Pro, wherein Zxy and Uvw independently each are any natural amino acid except Pro, whereby after the selective collagenase reaction, there is produced a protein with the sequence Gly-Pro-Zxy-Uvw-Pro at its N-terminal end. 10. The process of claim 9 wherein the amino acid Zxy is Met. 11. The process of claim 9 further comprising, subsequently selectively cleaving said Pro-Zxy-bond by treating the protein with postproline dipeptidylaminopeptidase, thereby producing a protein having the sequence Zxy-Uvw-Pro at its N-terminal end. 12. The process of claim 11 wherein the amino acid Zxy is Met. 13. The process of claim 11 further comprising selectively cleaving said Zxy residue from the resultant protein by treating the latter with leucine aminopeptidase. 14. The process of claim 13 wherein the amino acid Zxy is Met. 15. The process of claim 13 wherein the leucine aminopeptidase is E.C. 3.4.11.1. 16. The process of claim 15 wherein the amino acid Zxy is Met. 17. In a process for selectively cleaving a fusion protein produced from a genetically engineered microorganism containing a foreign gene, the fusion protein comprising native protein and foreign protein, the cleavage selectively occurring between the native and foreign protein, and the foreign protein being terminated at its N-terminal end by Pro, the improvement comprising incorporating the tripeptide sequence Pro-Xyz-Gly between the native and the foreign protein, wherein Xyz can be any natural amino acid and its carboxy end is attached to the amino end of Gly, and reacting the fusion protein with a collagenase whereby said Xyz-Gly bond is selectively cleaved, thereby producing a foreign protein with the sequence Gly-Pro at its N-terminal end. 18. The process of claim 17 wherein the collagenase is E.C. 3.4.24.3 (Clostridiopeptidase A). 19. The process of claim 17 further comprising subsequently selectively cleaving said glycine residue from the N-terminal end of the produced protein with an aminoacylproline aminopeptidase. 20. The process of claim 19 wherein the collagenase is E.C. 3.4.24.3, and the aminoacylproline aminopeptidase is aminopeptidase P, E.C. 3.4.11.9. 21. In a process for selectively cleaving Met from a foreign protein produced from a genetically engineered microorganism containing a foreign gene using the direct synthesis method wherein first the codon for Met is incorporated at the terminus of the foreign gene, whereby after culturing the microorganism, a foreign protein is obtained having Met attached to its N-terminal end, the improvement comprising first incorporating into the foreign gene the codons for Met-Pro attached to the N-terminal end of the subsequently produced foreign protein instead of only Met, whereby after culturing the microorganism Met-Pro is attached to the N-terminal end of the obtained foreign protein, and subsequently, selectively enzymatically cleaving Met-Pro to produce the foreign protein per se by treatment with postproline dipeptidylaminopeptidase. 22. In a process for selectively cleaving Met from a foreign protein produced from a genetically engineered microorganism containing a foreign gene using the direct synthesis method wherein first the codon for Met is incorporated at the terminus of the foreign gene, whereby after culturing the microorganism, a foreign protein is obtained having Met attached to its N-terminal end, and wherein the foreign protein per se contains the sequence Uvw-Pro- at its N-terminal end, the improvement comprising first incorporating into the foreign gene the codons for Met-Pro-Zxy attached to the N-terminal end of the subsequently produced foreign protein instead of only Met, whereby after culturing the microorganism, Met-Pro-Zxy is attached to the N-terminal end of the obtained foreign protein, Uvw and Zxy being any natural amino acid except Pro, and subsequently, selectively enzymatically cleaving Met-Pro from the N-terminal end by treatment with postproline dipeptidylaminopeptidase thereby producing a foreign protein having Zxy at its N-terminal end, and then selectively cleaving Zxy by treatment with leucine aminopeptidase to produce the foreign protein per se. 23. In a process for selectively cleaving Met from a foreign protein produced from a genetically engineered microorganism containing a foreign gene using the direct synthesis method wherein first the codon for Met is incorporated at the terminus of the foreign gene, whereby after culturing the microorganism, a foreign protein is obtained having Met attached to its N-terminal end, the improvement comprising first incorporating into the foreign gene the codons for Met-Pro attached to the N-terminal end of the subsequently produced foreign protein instead of only Met, whereby after culturing the microorganism, Met-Pro- is attached to the N-terminal end of the obtained foreign protein, and subsequently, selectively enzymatically cleaving Met from the N-terminal end by treatment with amino-acylproline aminopeptidase thereby producing a foreign protein having Pro at its N-terminal end, and then selectively cleaving Pro by treatment with proline aminopeptidase to produce the foreign protein per se.

Details for Patent 4,543,329

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2002-09-24