Claims for Patent: 6,017,762

Summary for Patent: 6,017,762

Title:	Cassette for the expression of an endothiapepsin precursor in Cryphonectria parasitica
Abstract:	The present invention relates to a new cassette for the expression of an endothiapepsin precursor in Cryphonectria parasitica, to a strain of this species transformed with this cassette, to a process for preparing endothiapepsin using this strain and also to a process for preparing such a strain devoid of a dominant selection marker.
Inventor(s):	Jara; Patrick (Castanet-Tolosan, FR), Legoux; Richard (Caraman, FR), Loison; Gerard (Toulouse, FR), Razanamparany; Voahangy (Toulouse, FR)
Assignee:	Sanofi (Paris, FR) Elf Aquitaine (Courbevoie, FR)
Application Number:	08/115,753
Patent Claims:	1. An endothiapepsin-producing Cryphonectria parasitica strain, wherein said strain is transformed with a cassette for the expression of an endothiapepsin precursor of Cryphonectria parasitica, said cassette comprising a functional promoter upstream of a nucleic acid sequence coding for said precursor, wherein endothiapepsin has the following amino acid sequence (PI) (SEQ ID NO:1), wherein said precursor is preproendothiapepsin comprising the following amino acid sequence (P4) (SEQ ID NO:2): 2. The strain of claim 1, wherein the nucleic acid sequence encoding said preproendothiapepsin comprises the following nucleic acid sequence (N4a) (SEQ ID NO:3): 3. The strain of claim 11, wherein said promoter is derived from a filamentous fungus of the class Ascomycetes. 4. The strain of claim 1, wherein said strain is devoid of a dominant selection marker. 5. The transformed strain of claim 1, wherein the untransformed Cryphonectria parasitica is the strain SEBR103, deposited with CNCM on Aug. 31, 1990, under no.degree. I-997. 6. The strain of claim 2, wherein the nucleic acid sequence N4a (SEQ ID NO:3) is interrupted by at least one intron. 7. The strain of claim 6, wherein the nucleic acid sequence encoding said preproendothiapepsin comprises the following nucleic acid sequence (N4b) (SEQ ID NO:4): 8. The strain of claim 3, wherein said promoter is a Cryphonectria parasitica gene promoter. 9. The strain of claim 3, wherein said promoter is the glyceraldehyde-3-phosphate dehydrogenase promoter of Aspergillus nidulans. 10. The strain of claim 8, wherein said promoter is the preproendothiapepsin Cryphonectria parasitica promoter. 11. The strain of claim 10, wherein the functional portion of said promoter is the 5'-Bgl II to Sca I segment of fragment C of FIG. 4. 12. The strain of claim 10, wherein the functional portion of said promoter is the 5'-Bam HI to Sca I segment of fragment C of FIG. 4. 13. The strain of claim 10, wherein said promoter comprises a sequence of the following nucleic acid sequence (N5) (SEQ ID NO:5): which carries the TATA box and, upstream of said sequence, a segment comprised of fragment C and bounded by the 5'-Bam HI site and the Hind III site at the 5'-end of fragment A of FIG. 4, such that said segment contains an activator region. 14. The strain of claim 13, wherein segment X of fragment C comprises the following nucleic acid sequence (SEQ ID NO:6): 15. The transformed strain according to claim 5, wherein said strain overproduces endothiapepsin when compared with SEBR103 by a factor of at least two. 16. A process for preparing endothiapepsin, wherein said process comprises (a) culturing an endothiapepsin-producing Cryphonectria parasitica strain, wherein said strain is transformed with a cassette for the expression of an endothiapepsin precursor of Cryphonectria parasitica, said cassette comprising a functional promoter upstream of a nucleic acid sequence coding for said precursor, wherein said precursor has the following amino acid sequence (P4) (SEO ID NO:2): and wherein said strain overproduces endothiapepsin compared with Cryphonectria parasitica, followed by (b) isolation and purification of said endothiapepsin. 17. A process for obtaining an endothiapepsin-producing Cryphonectria parasitica strain, wherein said strain is transformed with a cassette for the expression of an endothiapepsin precursor of Cryphonectria parasitica, said cassette comprising a functional promoter upstream of a nucleic acid sequence coding for said precursor, wherein endothiapepsin has the following amino acid sequence (PI) (SEQ ID NO:1): and wherein said strain overproduces endothiapepsin compared with Cryphonectria parasitica, comprising (a) at least one cycle of cotransformation of mycelium protoplasts with said cassette and a dominant selection marker, followed by (b) purification by sporulation wherein said marker is removed. 18. The process of claim 17, wherein said process comprises two cycles of cotransformation before said purification.

Details for Patent 6,017,762

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2017-01-25
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2017-01-25
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2017-01-25