IP Library › Granted Patent US 8,148,508
Granted Patent B2
US 8,148,508 · App. 12/861,714 · Granted Apr 3, 2012

Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides

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Quick Facts
Patent No.
US 8,148,508
App. No.
12/861,714
Granted
Apr 3, 2012
Kind
B2
Abstract

The invention provides methods for making peptides from a polypeptide containing at least one copy of the peptide using clostripain to excise the peptide from the polypeptide. The methods enable the use of a single, highly efficient enzymatic cleavage to produce any desired peptide sequence.

Claims (53)

1. An isolated nucleic acid encoding a polypeptide for production of a GLP-2 peptide, the encoded polypeptide comprising:

two or more copies of the GLP-2 peptide; and

a clostripain cleavage recognition sequence;

wherein the clostripain cleavage recognition sequence is comprised of amino acids at the N-terminus and/or the C-terminus of the GLP-2 peptide and/or of an optional linker, the clostripain cleavage recognition sequence positioned to allow cleavage by clostripain between the copies of the GLP-2 peptide;

wherein the clostripain cleavage recognition sequence comprises Formula I:

-Xaa 1 -Xaa 2 -Xaa 3 -  (I);

wherein Xaa 1 is aspartic acid, glycine, proline or glutamic acid, Xaa 2 is arginine, and Xaa 3 is not an acidic amino acid.

2. The nucleic acid of claim 1 , wherein the GLP-2 peptide is a native GLP-2 peptide.

3. The nucleic acid of claim 2 , wherein the GLP-2 peptide is GLP-2(1-34) (SEQ ID NO:9).

4. The nucleic acid of claim 2 , wherein the GLP-2 peptide is GLP-2(1-33) (SEQ ID NO:11).

5. The nucleic acid of claim 1 , wherein the GLP-2 peptide is a variant of a native GLP-2 peptide.

6. The nucleic acid of claim 5 , wherein the GLP-2 peptide is GLP-2(1-34,A2G) (SEQ ID NO:15).

7. The nucleic acid of claim 5 , wherein the GLP-2 peptide is GLP-2(1-33,A2G) (SEQ ID NO:13).

8. The nucleic acid of claim 1 , wherein the encoded polypeptide for production of the GLP-2 peptide comprises the following formula:

Tag-Linker-[Peptide] q

wherein,

Tag comprises SEQ ID NO:17 or 18;

Linker comprises at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;

Peptide is GLP-2(1-34) (SEQ ID NO: 9) or GLP-2(1-34, A2G) (SEQ ID NO: 15); and

q is an integer of 2 to about 20.

9. The nucleic acid of claim 1 , wherein the encoded polypeptide for production of the GLP-2 peptide comprises the following formula:

Tag-Linker-[Peptide-Linker 2 ] q

wherein:

Tag comprises SEQ ID NO:17 or SEQ ID NO: 18;

Linker comprises at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;

Linker 2 is SEQ ID NO:23;

Peptide is GLP-2(1-34) (SEQ ID NO: 9) or GLP-2(1-34, A2G) (SEQ ID NO: 15); and

q is an integer of 2 to about 20.

10. An isolated nucleic acid encoding a polypeptide for production of a GLP-2 peptide, the encoded polypeptide comprising:

a GLP-2 peptide;

wherein the GLP-2 peptide is selected from GLP-2(1-34) (SEQ ID NO:9), GLP-2(1-33) (SEQ ID NO:11), GLP-2(1-34,A2G) (SEQ ID NO:15) and GLP-2(1-33,A2G) (SEQ ID NO:13); and

a clostripain cleavage recognition sequence;

wherein the clostripain cleavage recognition sequence is comprised of amino acids at the N-terminus of the GLP-2 peptide and/or of an optional linker, the clostripain cleavage recognition sequence positioned to allow cleavage by clostripain at the N-terminus of the GLP-2 peptide;

wherein the clostripain cleavage recognition sequence comprises Formula I:

-Xaa 1 -Xaa 2 -Xaa 3 -  (I);

wherein Xaa 1 is aspartic acid, glycine, proline or glutamic acid, Xaa 2 is arginine, and Xaa 3 is not an acidic amino acid.

11. The nucleic acid of claim 10 , wherein the GLP-2 peptide is a native GLP-2 peptide.

12. The nucleic acid of claim 11 , wherein the GLP-2 peptide is GLP-2(1-34) (SEQ ID NO:9).

13. The nucleic acid of claim 11 , wherein the GLP-2 peptide is GLP-2(1-33) (SEQ ID NO:11).

14. The nucleic acid of claim 10 , wherein the GLP-2 peptide is a variant of a native GLP-2 peptide.

15. The nucleic acid of claim 14 , wherein the GLP-2 peptide is GLP-2(1-34,A2G) (SEQ ID NO:15).

16. The nucleic acid of claim 14 , wherein the GLP-2 peptide is GLP-2(1-33,A2G) (SEQ ID NO:13).

17. The nucleic acid of claim 10 , wherein the encoded polypeptide for production of the GLP-2 peptide comprises the following formula:

Tag-Linker-Peptide

wherein,

Tag comprises SEQ ID NO:17 or 18;

Linker comprises at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; and

Peptide is GLP-2(1-33) (SEQ ID NO:11) or GLP-2(1-33,A2G) (SEQ ID NO:13).

18. An isolated nucleic acid encoding a polypeptide for production of a GLP-2 peptide, the encoded polypeptide comprising the following formula:

Xaa 1 -Xaa 2 -Peptide

wherein Xaa 1 is aspartic acid, glycine, proline or glutamic acid, Xaa 2 is arginine, and Peptide is a GLP-2 peptide; and

wherein the GLP-2 peptide is selected from GLP-2(1-34) (SEQ ID NO:9, GLP-2(1-33) (SEQ ID NO:11), GLP-2(1-34,A2G) (SEQ ID NO:15) and GLP-2(1-33,A2G) (SEQ ID NO:13).

19. The nucleic acid of claim 18 , wherein the GLP-2 peptide is GLP-2(1-33,A2G).