Fungal proteins and nucleic acids encoding same

• US 7,354,734 B2
• Filed: 08/25/2004
• Issued: 04/08/2008
• Est. Priority Date: 08/25/2003
• Status: Active Grant

• Current Assignee: AMYRA Biotech AG
• Original Assignee: Funzyme Biotechnologies SA
• Inventors: Grouzmann, Eric, Stocklin, Reto, Monod, Michel
• Primary Examiner(s): Slobodyansky; Elizabeth
• Application Number:
  US10/926,188
• Publication Number:
  US 20050158298A1
• Time in Patent Office: 1,322 Days
• Field of Search: None
• US Class Current: 435/68.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/37 from fungi
• CPC Class Codes: C12N 9/48 acting on peptide bonds (3.4)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/37 from fungi

Abstract

Disclosed herein are fungal nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The novel leucine aminopeptidase (LAP) and other amino- and carboxypeptidases polypeptides, referred to herein as EXOX nucleic acids and proteins of the invention are useful in a variety of medical, research, and commercial applications.

Claims

1. A method of degrading a polypeptide substrate, the method comprising contacting the polypeptide substrate with a leucine aminopeptidase, wherein the leucine aminopeptidase comprises an amino acid sequence which is at least 95% identical to SEQ ID NO:

3, or a fragment thereof, wherein said fragment has leucine aminopeptidase activity.

2. The method of claim 1, wherein the polypeptide substrate is a full-length protein.

3. The method of claim 1, wherein the leucine aminopeptidase sequentially digests the polypeptide substrate.

4. The method of claim 1, wherein the polypeptide substrate is denatured casein, or fragments thereof.

5. The method of claim 1, wherein the leucine aminopeptidase comprises the amino acid sequence set forth as SEQ ID NO:

3.

6. The method of claim 1, further comprising contacting the polypeptide substrate with one or more proteases.

7. The method of claim 6, wherein said one or more protease is trypsin.

13. The method of claim 1, comprising contacting the polypeptide substrate with a dipeptidyl peptidase IV (DPPIV), wherein the DPPIV comprises an amino acid sequence which is at least 95% identical to SEQ ID NO:

35, or a fragment thereof, wherein said fragment has IV dipeptidyl peptidase activity.

14. The method of claim 13, wherein the DPPIV comprises the amino acid sequence set forth in SEQ ID NO;

35.

15. The method of claim 13, wherein the DPPIV consists of the amino acid sequence set forth in SEQ ID NO:

35.

16. The method of claim 1, wherein the leucine aminopeptidase comprises an amino acid sequence which is at least 98% identical to SEQ ID NO:

3.

17. The method of claim 1, wherein the leucine aminopeptidase comprises the amino acid sequence set forth as SEQ ID NO:

33.

18. The method of claim 1, wherein the leucine aminopeptidase comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

3.

19. The method of claim 18, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) asparic acid or glutamic acid for each other;

iii) glycine, asparagine, glutamine, serine, threonine, tyrosine. or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valine, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

22. The method of claim 1, wherein the leucine aminopeptidase consists of the amino acid sequence set forth as SEQ ID NO:

3.

23. The method of claim 1, wherein the polypeptide substrate is gliadin, or fragments thereof.

24. The method of claim 1, wherein the polypeptide substrate is gluten, or fragments thereof.

25. The method of claim 1, wherein the polypeptide substrate is bovine serum albumin, or fragments thereof.

26. The method of claim 6, wherein said one or more protease is pronase.

27. The method of claim 6, wherein said one or more protease is chymotrypsin.

28. The method of claim 6, wherein said one or more prorease is proteinase K.

8. A method of removing amino acids from the amino terminus of a polypeptide substrate comprising an amino terminus tag, comprising contacting the polypeptide substrate comprising the amino terminus tag with a leucine aminopeptidase, wherein the leucine aminopeptidase comprises an amino acid sequence which is at least 95% identical to SEQ ID NO:

3, or a fragment thereof, wherein said fragment has leucine aminopeptidase activity.

9. The method of claim 8, wherein said amino terminus tag comprises a His tag.

10. The method of claim 8, wherein said amino terminus tag comprises Xaa-Pro.

11. The method of claim 10, wherein Xaa is an amino acid comprising at least two vicinal nucleophilic groups.

12. The method of claim 11, wherein the amino acid is serine, threonine or cysteine.

20. The method of claim 8, wherein the leucine aminopeptidase comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

3.

21. The method of claim 20, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) aspartic acid or glutanic acid for each other;

iii) glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valline, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

29. The method of claim 8, wherein the leucine aminopeptidase comprises the amino acid sequence set forth as SEQ ID NO:

3.

30. The method of claim 8, wherein the leucine aminopeptidase consists of the amino acid sequence set forth as SEQ ID NO. 3.

31. The method of claim 8, wherein the leucine aminopeptidase comprises an amino acid sequence which is at least 98% identical to SEQ ID NO:

3.

32. The method of claim 8, wherein the leucine aminopeptidase comprises the amino acid sequence set forth as SEQ ID NO:

33.