Claims for Patent: 5,679,543

Summary for Patent: 5,679,543

Title:	DNA sequences, vectors and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi
Abstract:	The invention includes novel fusion DNA sequences encoding fusion polypeptides which when expressed in a filamentous fungus result in increased levels of secretion of the desired polypeptide as compared to the expression and secretion of such polypeptides from filamentous fungi transformed with previously used DNA sequences. The fusion DNA sequences comprise from the 5\' terminus four DNA sequences which encode a fusion polypeptide comprising, from the amino to carbonyl-terminus, first, second, third and fourth amino acid sequences. The first DNA sequence encodes a signal peptide functional as a secretory sequence in a first filamentous fungus. The second DNA sequence encodes a secreted polypeptide or portion thereof which is normally secreted from the same filamentous fungus or a second filamentous fungus. The third DNA sequence encodes a cleavable linker polypeptide while the fourth DNA sequence encodes a desired polypeptide. When the fusion DNA sequence is expressed either in the first or second filamentous fungus, increased secretion of the desired polypeptide is obtained as compared to that which is obtained when the desired polypeptide is expressed from DNA sequences encoding a fusion polypeptide which does not contain the second polypeptide normally secreted from either of the filamentous fungi.
Inventor(s):	Lawlis; Virgil Bryan (San Mateo, CA)
Assignee:	Genencor International, Inc. (Rochester, NY)
Application Number:	08/318,491
Patent Claims:	1. A fusion DNA sequence encoding a fusion polypeptide comprising, from the 5' end of said fusion DNA sequence, first, second, third and fourth DNA sequences encoding, from the amino- to carboxy-terminus of said fusion polypeptide, corresponding first, second, third and fourth amino acid sequences, said first DNA sequence encoding a signal peptide functional as a secretory sequence in a first filamentous fungus, said second DNA sequence encoding a mature form of a secreted polypeptide normally secreted from said first or a second filamentous fungus or portion thereof comprising greater than 50% of the amino terminal sequence of said secreted polypeptide, said third DNA sequence encoding a cleavable linker polypeptide and said fourth DNA sequence encoding a desired polypeptide, wherein said first and said second filamentous fungi are selected from the group consisting of Aspergillus, Trichoderma and Neurospora and the expression of said fusion DNA sequence in said first or said second filamentous fungus results in increased secretion of said desired polypeptide as compared to the secretion of said desired polypeptide from said first or said second filamentous fungus when expressed as a second fusion polypeptide encoded by a second fusion DNA sequence comprising only said first, third and fourth DNA sequences. 2. The fusion DNA sequence of claim 1 wherein said first DNA sequence encodes a signal peptide or portion thereof selected from the group consisting of signal peptides from glucoamylase, .alpha.-amylase, and aspartyl protease from Aspergillus spp., signal peptides from bovine chymosin and human tissue plasminogen activator and signal peptides from Trichoderma cellobiohydrolase I and II. 3. The fusion DNA sequence of claim 1 wherein said first DNA sequence encodes the signal peptide from Aspergillus awamori glucoamylase. 4. The fusion DNA sequence of claim 1 wherein said second DNA sequence encodes a secreted polypeptide selected from the group consisting of glucoamylase, .alpha.-amylase, and aspartyl protease from Aspergillus spp. and Trichoderma cellobiohydrolase I and II. 5. The fusion DNA sequence of claim 1 wherein said second DNA sequence encodes glucoamylase from Aspergillus awamori. 6. The fusion DNA sequence of claim 1 wherein said third DNA sequence encodes a cleavable linker polypeptide selected from the group consisting of the prosequence from chymosin, the prosequence of subtilisin, and sequences recognized by trypsin factor X.sub.a, collagenase, clostripain, subtilisin and chymosin. 7. The DNA sequence of claim 1 wherein said third DNA sequence encodes the prosequence of chymosin or a portion thereof. 8. The fusion DNA sequence of claim 1 wherein said fourth DNA sequence encodes a desired polypeptide selected from the group consisting of enzymes, proteinaceous hormones and serum proteins. 9. The fusion DNA sequence of claim 1 wherein said fourth DNA sequence encodes bovine chymosin. 10. The fusion DNA sequence of claim 1 wherein said first DNA sequence encodes the signal peptide from Aspergillus awamori glucoamylase, said second sequence encodes glucoamylase from Aspergillus awamori said third sequence encodes the prosequence of chymosin and said fourth sequence encodes chymosin. 11. An expression vector for transforming a host filamentous fungus selected from the group consisting of Aspergillus, Trichoderma and Neurospora comprising DNA sequences 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 DNA sequence of claim 1 and transcription stop sequences and polyadenylation sequences operably linked to the 3' end of said fusion DNA sequence. 12. The expression vector of claim 11 wherein said first and said second DNA sequences encoding respectively said signal peptide and said secreted polypeptide are selected from filamentous fungi of the same genus as said host filamentous fungus. 13. The expression vector of claim 12 wherein said genus is selected from the group consisting of Aspergillus, Trichoderma, and Neurospora. 14. The expression vector of claim 12 wherein said genus is Aspergillus. 15. The expression vector of claim 11 wherein said first and said second DNA sequences encoding respectively said signal peptide and said secreted polypeptide are from said host filamentous fungus. 16. A host filamentous fungus selected from the group consisting of Aspergillus, Trichoderma and Neurospora comprising any one of the expression vectors of claims 11 through 15. 17. A fusion polypeptide comprising, from the amino-to carboxy-terminus, first, second, third and fourth amino acid sequences, said first amino acid sequence comprising a signal peptide functional as a secretory sequence in a first filamentous fungus, said second amino acid sequence comprising a mature form of a secreted polypeptide normally secreted from said first or a second filamentous fungus or portion thereof comprising greater than 50% of the amino terminal sequence of said secreted polypeptide, said third amino acid sequence comprising a cleavable linker polypeptide and said fourth amino acid sequence comprising a desired polypeptide, wherein said first and said second filamentous fungi are selected from the group consisting of Aspergillus, Trichoderma and Neurospora and the expression of the fusion DNA sequence encoding said fusion polypeptide in said first or said second filamentous fungus results in increased secretion of said desired polypeptide as compared to the secretion of said desired polypeptide from said first or said second filamentous fungus when expressed from a second fusion DNA sequence encoding a second fusion polypeptide comprising said first, third and fourth amino acid sequences. 18. The fusion polypeptide of claim 17 wherein said first amino acid sequence comprises a signal peptide or portion thereof selected from the group consisting of signal peptides from glucoamylase, .alpha.-amylase, and aspartyl protease from Aspergillus spp., signal peptides from bovine chymosin and human tissue plasminogen activator and signal peptides from Trichoderma cellobiohydrolase I and II. 19. The fusion polypeptide of claim 17 wherein said first amino acid sequence is the signal peptide from Aspergillus awamori glucoamylase. 20. The fusion polypeptide of claim 17 wherein said second amino acid sequence is selected from the group consisting of glucoamylase, .alpha.-amylase, and aspartyl protease from Aspergillus spp. and Trichoderma cellobiohydrolase I and II. 21. The fusion polypeptide of claim 17 wherein said second amino acid sequence is glucoamylase from Aspergillus awamori. 22. The fusion polypeptide of claim 17 wherein said cleavable linker polypeptide is selected from the group consisting of the prosequence of subtilisin, and sequences recognized by trypsin Factor Xa, collagenase, clostripain, subtilisin and chymosin. 23. The fusion polypeptide of claim 17 wherein said third amino acid sequence is the prosequence of chymosin. 24. The fusion polypeptide of claim 17 wherein said fourth amino acid sequence is selected from the group consisting of enzymes, proteinaceous hormones and serum proteins. 25. The fusion polypeptide of claim 17 wherein said fourth amino acid sequence is chymosin. 26. The fusion polypeptide of claim 17 wherein said first amino acid sequence is the signal peptide of A. awamori glucoamylase, said second amino acid sequence is glucoamylase from A. awamori said third amino acid sequence is the prosequence of chymosin and said fourth amino acid sequence is bovine chymosin. 27. A process for producing a desired polypeptide comprising: transforming a host filamentous fungus selected from the group consisting of Aspergillus, Trichoderma and Neurospora with an expression vector containing the vector of claim 11 under conditions which permit expression of said fusion DNA sequence to cause the secretion of the desired polypeptide encoded by said fusion DNA sequence. 28. The fusion DNA of claim 1 wherein said first and said second filamentous fungi are from the same genus. 29. The fusion DNA of claim 28 wherein said genus is selected from the group consisting of Aspergillus, Trichoderma, and Neurospora. 30. The fusion DNA of claim 28 wherein said genus comprises Aspergillus. 31. The fusion polypeptide of claim 17 wherein said first and said second filamentous fungi are from the same genus. 32. The fusion polypeptide of claim 31 wherein said genus is selected from the group consisting of Aspergillus, Trichoderma, and Neurospora. 33. The fusion polypeptide of claim 31 wherein said genus comprises Aspergillus. 34. The fusion DNA sequence of claim 1 wherein said portion comprises greater than 75% of the amino acid sequence of said secreted polypeptide. 35. The fusion DNA of claim 1 wherein said portion comprises greater than 90% of the amino acid sequence of said secreted polypeptide. 36. The fusion DNA of claims 1, 34 or 35 wherein said portion of said secreted polypeptide is the amino terminal portion. 37. The fusion polypeptide of claim 17 wherein said portion comprises greater than 75% of the amino acid sequence of said secreted polypeptide. 38. The fusion polypeptide of claim 17 wherein said portion comprises greater than 90% of the amino terminal portion of said secreted polypeptide. 39. The fusion polypeptide of claim 17, 37 or 38 wherein said portion of said secreted polypeptide is the amino terminal portion. 40. A fusion DNA sequence encoding a fusion polypeptide comprising, from the 5' end of said fusion DNA sequence, first, second, third and fourth DNA sequences encoding, from the amino- to carboxy-terminus of said fusion polypeptide, corresponding first, second, third and fourth amino acid sequences, wherein a) said first DNA sequence encodes a signal peptide functional as a secretory sequence in a first filamentous fungus; b) said second DNA sequence encodes a mature form of a secreted polypeptide normally secreted from said first or a second filamentous fungus or portion thereof comprising greater than 50% of the amino terminal sequence of said secreted polypeptide, and is selected from the group consisting of glucoamylase, .alpha.-amylase and aspartyl proteases from Aspergillus awamori, Aspergillus niger, and Aspergillus oryzae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, and endoglucanase III sequences from Trichoderma, and glucoamylase sequences from Neurospora and Humicola; c) said third DNA sequence encodes a cleavable linker polypeptide; and d) said fourth DNA sequence encodes a desired polypeptide; wherein said first and said second filamentous fungi are selected from the group consisting of Aspergillus, Trichoderma and Neurospora and the expression of said fusion DNA sequence in said first or said second filamentous fungus results in increased secretion of said desired polypeptide as compared to the secretion of said desired polypeptide from said first or said second filamentous fungus when expressed as a second fusion polypeptide encoded by a second fusion DNA sequence comprising only said first, third and fourth DNA sequences. 41. A fusion DNA sequence according to claim 40 wherein said first DNA sequence is selected from the group consisting of glucoamylase, .alpha.-amylase and aspartyl protease signal sequences from Aspergillus awamori, Aspergillus niger, and Aspergillus oryzae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, and endoglucanase III signal sequences from Trichoderma, glucoamylase signal sequences from Neurospora and Humicola, bovine chymosin signal sequence, human tissue plasminogen activator signal sequence, human interferon signal sequence, and synthetic consensus eukaryotic signal sequences. 42. A fusion DNA sequence according to claim 44 herein said third DNA sequence is selected from the group consisting of the prosequence of bovine chymosin, the prosequence of subtilisin, the prosequence of human immunodeficiency virus protease, and sequences recognized and cleaved by trypsin, Factor Xa, collagenase, clostripin, subtilisin, chymosin, and yeast KEX2 protease. 43. A fusion DNA sequence encoding a fusion polypeptide according to claim 42 wherein said desired polypeptide is selected from the group consisting of bovine chymosin, human tissue plasminogen activator, human growth hormone, human interferon, human interleukin, human serum albumin, Bacillus .alpha.-amylase, Pseudomonas lipase, lignin peroxidase and Mn2+-dependent peroxidase from Phanerochaete, Humicola glucoamylase, and Mucor aspartyl proteases. 44. A fusion polypeptide comprising, from the amino-to carboxy-terminus, first, second, third and fourth amino acid sequences, wherein: a) said first amino acid sequence comprises a signal peptide functional as a secretory sequence in a first filamentous fungus; b) said second amino acid sequence comprises a mature form of a secreted polypeptide normally secreted from said first or a second filamentous fungus or portion thereof comprising greater than 50% of the amino terminal sequence of said secreted polypeptide, and is selected from the group consisting of glucoamylase, .alpha.-amylase and aspartyl proteases from Aspergillus awamori, Aspergillus niger, and Aspergillus oryzae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, and endoglucanase III sequences from Trichoderma, and glucoamylase sequences from Neurospora and Humicola; c) said third amino acid sequence comprises a cleavable linker polypeptide; and d) said fourth amino acid sequence comprises a desired polypeptide; wherein said first and said second filamentous fungi are selected from the group consisting of Aspergillus, Trichoderma and Neurospora and the expression of the fusion DNA sequence encoding said fusion polypeptide in said first or said second filamentous fungus results in increased secretion of said desired polypeptide as compared to the secretion of said desired polypeptide from said first or said second filamentous fungus when expressed from a second fusion DNA sequence encoding a second fusion polypeptide comprising said first, third and fourth amino acid sequences. 45. A fusion polypeptide according to claim 44 wherein said first amino acid sequence is selected from the group consisting of glucoamylase, .alpha.-amylase and aspartyl protease signal sequences from Aspergillus awamori, Aspergillus niger, and Aspergillus oryzae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, and endoglucanase III signal sequences from Trichoderma, glucoamylase signal sequences from Neurospora and Humicola, bovine chymosin signal sequence, human tissue plasminogen activator signal sequence, human interferon signal sequence, and synthetic consensus eukaryotic signal sequences. 46. A fusion polypeptide according to claim 45 wherein said first amino acid sequence is selected from the group consisting of the prosequence of bovine chymosin, the prosequence of subtilisin, the prosequence of human immunodeficiency virus protease, and sequences recognized and cleaved by trypsin, Factor Xa, collagenase, clostripin, subtilisin, chymosin, and yeast KEX2 protease. 47. A fusion polypeptide according to claim 46 wherein said desired polypeptide is selected from the group consisting of bovine chymosin, human tissue plasminogen activator, human growth hormone, human interferon, human interleukin, human serum albumin, Bacillus .alpha.-amylase, Pseudomonas lipase, lignin peroxidase and Mn2+-dependent peroxidase from Phanerochaete, Humicola glucoamylase, and Mucor aspartyl proteases.

Details for Patent 5,679,543

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2014-10-21