Fungal dipeptidyl peptidase IV (DPPIV) enzyme for reagent use

• US 7,943,340 B2
• Filed: 07/03/2008
• Issued: 05/17/2011
• 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): Swope; Sheridan
• Application Number:
  US12/217,470
• Publication Number:
  US 20090130737A1
• Time in Patent Office: 1,048 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 dipetidyl peptidase IV (DPPIV) 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 aminopeptidase polypeptides, referred to herein as DPPIV proteins of the invention are useful in a variety of medical, research, and commercial applications.

Claims

1. A method for removing an aminoterminal Xaa-Pro tag from a protein, wherein Xaa is any amino acid, comprising the steps of:

(1) purification of the protein by covalent capture, and,

(2) contacting the protein with a polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO;

35, or a fragment thereof, wherein said polypeptide or fragment has dipeptidyl peptidase IV activity, wherein the DPPIV activity consists of cleavage of the aminoterminal Xaa-Pro tag of the protein such that the aminoterminal Xaa-Pro is removed.

2. The method of claim 1, wherein Xaa is an amino acid comprising at least two vicinal nucleophilic groups.

3. The method of claim 2, wherein said amino acid is selected from the group consisting of serine, threonine and cysteine.

4. The method of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

35.

5. The method of claim 1, wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:

35.

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

35.

7. The method of claim 3, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

35.

8. The method of claim 3, wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:

35.

9. The method of claim 3, wherein the polypeptide comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

35.

10. A method of removing the amino terminus from a protein wherein the amino terminus consists of an aminoterminal Xaa-Pro tag, wherein Xaa is any amino acid, said method comprising contacting the protein with polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO:

35, or a fragment thereof, wherein said polypeptide or fragment has dipeptidyl peptidase IV (DPPIV) activity, wherein said DPPIV activity consists of cleaveage of the aminoterminal Xaa-Pro tag, such that the amino terminus of the protein is removed.

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 said amino acid is selected from the group consisting of serine, threonine and cysteine.

13. The method of claim 10, wherein the DPPIV polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

35.

14. The method of claim 10, wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:

35.

15. The method of claim 10, wherein the polypeptide comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

35.

16. The method of claim 12, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

35.

17. The method of claim 12,wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:

35.

18. The method of claim 12, wherein the polypeptide comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

35.