Claims for Patent: 6,265,204

Summary for Patent: 6,265,204

Title:	DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
Abstract:	The invention includes novel fusion nucleic acids encoding fusion polypeptides which when expressed in a filamentous fungus result in the expression of fusion polypeptides. The fusion nucleic acids comprise four nucleic acids which encode a fusion polypeptide comprising first, second, third and fourth amino acid sequences. The first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus. The second nucleic acid encodes a secreted polypeptide or functional portion thereof which is normally secreted from the same filamentous fungus or a second filamentous fungus. The third nucleic acid encodes a cleavable linker while the fourth nucleic acid comprises at least two nucleic acids encoding desired polypeptides.
Inventor(s):	Ward; Michael (San Francisco, CA), Power; Scott D. (San Bruno, CA)
Assignee:	Genencor International, Inc. (Rochester, NY)
Application Number:	08/785,668
Patent Claims:	1. A fusion nucleic acid encoding a fusion polypeptide comprising, from a 5' end of said fusion nucleic acid, first, second, third and fourth nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide or functional portion thereof from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fourth nucleic acid comprises two or more nucleic acids each encoding desired polypeptides. 2. A fusion nucleic acid according to claim 1 wherein said two or more nucleic acids encode the same polypeptide. 3. A fusion nucleic acid according to claim 1 wherein said fourth nucleic acid comprises at least two nucleic acids which encode different desired polypeptides. 4. A fusion nucleic acid according to claim 1 wherein said fourth nucleic acid further comprises at least one nucleic acid encoding a cleavable linker, wherein said nucleic acids encoding said desired polypeptides are separated by said nucleic acid encoding said cleavable linker. 5. A fusion nucleic acid according to claim 4 wherein said fourth nucleic acid comprises at least three nucleic acids encoding desired polypeptides and at least two nucleic acids encoding cleavable linkers, wherein each of said nucleic acids encoding desired polypeptides is separated by one of said nucleic acids encoding a cleavable linker. 6. A fusion nucleic acid according to claim 4 wherein said fourth nucleic acid comprises at least four nucleic acids encoding desired polypeptides and at least three nucleic acids encoding cleavable linkers, wherein each of said nucleic acids encoding desired polypeptides is separated by one of said nucleic acids encoding a cleavable linker. 7. A fusion nucleic acid according to claim 5 wherein said fourth nucleic acid comprises at least four nucleic acids encoding desired polypeptides and at least three nucleic acids encoding cleavable linkers, wherein each of said nucleic acids encoding a cleavable linker is positioned between nucleic acids encoding desired polypeptides. 8. A fusion nucleic acid encoding a fusion polypeptide comprising, from a 5' end of said fusion nucleic acid, first, fourth, third and second nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide or functional portion thereof from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fifth nucleic acid comprises at least one nucleic acid encoding a desired polypeptide. 9. A fusion nucleic acid encoding a fusion polypeptide comprising: a) a first nucleic acid encoding a signal polypeptide functional as a secretory sequence in a first filamentous fungus; operably linked to b) a second nucleic acid comprising first and second parts together encoding a secreted polypeptide or functional portion thereof from said first or a second filamentous fungus; and c) an insertion nucleic acid between said first and second parts comprising a fourth nucleic acid flanked by third nucleic acids; wherein said third nucleic acids encode cleavable linkers and said fourth nucleic acid comprises at least one nucleic acid encoding a desired polypeptide. 10. A fusion nucleic acid encoding a fusion polypeptide comprising, from a 5' end of said fusion nucleic acid: a) a first nucleic acid encoding a signal polypeptide functional as a secretory sequence in a first filamentous fungus; operably linked to b) a second nucleic acid comprising first and second parts and encoding a secreted polypeptide or functional portion thereof from said first or a second filamentous fungus, wherein said secreted polypeptide contains a linker region; and c) an insertion nucleic acid between said first and second parts comprising a fourth nucleic acid flanked by third nucleic acids, wherein the insertion point is within said linker region; wherein said third nucleic acids encode cleavable linkers and said fifth nucleic acid comprises at least one nucleic acid encoding a desired polypeptide. 11. A fusion nucleic acid according to claim 1, 8 or 9 wherein said first nucleic acid encodes a signal polypeptide or portion thereof selected from the group consisting of signal polypeptides from glucoamylase, .alpha.-amylase, and aspartyl protease from Aspergillus species, signal polypeptides from bovine chymosin and human tissue plasminogen activator and signal polypeptides from Trichoderma cellobiohydrolase I and II. 12. A fusion nucleic acid according to claim 11 wherein said first nucleic acid encodes the signal polypeptide from Aspergillus awamori glucoamylase. 13. A fusion nucleic acid according to claim 1, 8 or 9 wherein said second nucleic acid encodes a secreted polypeptide selected from the group consisting of glucoamylase, .alpha.-amylase, and aspartyl protease from Aspergillus species and Trichoderma cellobiohydrolase I and II. 14. A fusion nucleic acid according to claim 13 wherein said second nucleic acid encodes at least the catalytic domain of glucoamylase from Aspergillus awamori. 15. A fusion nucleic acid according to claim 13 wherein said second nucleic acid encodes at least the catalytic domain and part of the linker region of glucoamylase from Aspergillus awamori. 16. A fusion nucleic acid according to claim 1, 8 or 9 wherein said second nucleic acid encodes glucoamylase from Aspergillus awamori. 17. A fusion nucleic acid according to claim 1, 8 or 9 wherein said third nucleic acid encodes a cleavable linker selected from the group consisting of the prosequence from chymosin, the prosequence of subtilisin, methionine, and sequences recognized by trypsin, factor X.sub.a, collagenase, clostripain, subtilisin and chymosin. 18. A fusion nucleic acid according to claim 1, 8 or 9 wherein said cleavable linker is a sequence recognized by trypsin. 19. A fusion nucleic acid according to claim 18 wherein said sequence is a lysine residue. 20. A fusion nucleic acid according to claim 18 wherein said sequence is lysine-arginine. 21. A fusion nucleic acid according to claim 1, 8 or 9 wherein said third nucleic acid encodes the prosequence of chymosin or a portion thereof. 22. A fusion nucleic acid according to claim 1, 8 or 9 wherein said fourth nucleic acid encodes a desired polypeptide selected from the group consisting of enzymes, hormones, growth factors, cytokines, and serum proteins. 23. A fusion nucleic acid according to claim 1, 8 or 9 wherein said fourth nucleic acid encodes R3-hirulog. 24. An expression vector for transforming a host filamentous fungus comprising nucleic acids encoding regulatory sequences functionally recognized by said host filamentous fungus including promoter and transcription and translation initiation sequences operably linked to the 5' end of the fusion nucleic acid of claim 1, 8 or 9 and transcription stop sequences and polyadenylation sequences operably linked to the 3' end of said fusion nucleic acid. 25. The expression vector of claim 24 wherein said first and said second nucleic acids encoding respectively said signal polypeptide and said secreted polypeptide are selected from filamentous fungi of the same genus as said host filamentous fungus. 26. The expression vector of claim 24 wherein said genus is selected from the group consisting of Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, Achlya, Fusarium and Humicola. 27. The expression vector of claim 26 wherein said genus is Aspergillus. 28. The expression vector of claim 24 wherein said first and said second nucleic acids encoding respectively said signal polypeptide and said secreted polypeptide are from said host filamentous fungus. 29. A filamentous fungus containing a fusion nucleic acid according to claim 1, 8 or 9. 30. A filamentous fungus containing an expression vector according to claim 24. 31. A process for producing a desired polypeptide comprising transforming a host filamentous fungus with an expression vector containing the fusion nucleic acid of claim 1, 8 or 9 under conditions which permit expression of said fusion nucleic acid to cause the secretion of the desired polypeptide encoded by said fusion nucleic acid. 32. A fusion nucleic acid encoding a fusion polypeptide comprising, from a 5' end of said fusion nucleic acid, first, second, third and fourth nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide normally secreted from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fourth nucleic acid comprises two or more nucleic acids each encoding desired polypeptides.

Details for Patent 6,265,204

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-02-26