IP Library › Patent Application 12354053
Patent Application
App. No. 12/354,053

METHOD FOR UNIVERSAL ENZYMATIC PRODUCTION OF BIOACTIVE PEPTIDES

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Quick Facts
Patent No.
US None
App. No.
12/354,053
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 (28)

1 - 2 . (canceled)

3 . A method for producing a desired peptide having a C-terminal acidic, aliphatic or aromatic amino acid from a polypeptide, comprising:

combining clostripain and the polypeptide comprising formula (III):

(Linker-Xaa 3 -Peptide 1 ) n -Linker-Xaa 3 -Peptide 1   (III);

wherein

the desired peptide comprises Xaa 3 -Peptide 1 ;

Peptide 1 is any amino acid sequence other than Xaa 1 -Xaa 2 ;

n is an integer ranging from 0 to 50;

Xaa 3 is not an acidic amino acid;

Linker is a cleavable peptide linker having Formula (IV):

(Peptide 5 ) m —Xaa 1 -Xaa 2   (IV);

wherein

m is an integer ranging from 0 to 50;

Xaa 1 is aspartic acid, glycine, proline or glutamic acid;

Xaa 2 is arginine; and

Peptide 5 is any single amino acid residue or amino acid sequence not containing Xaa 1 -Xaa 2 .

4 . The method of claim 1 , wherein the polypeptide is a soluble polypeptide.

5 . The method of claim 1 , wherein the cleavage is performed at about 18° C. to about 25° C.

6 . The method of claim 1 , wherein the cleavage is performed between a pH of about 5 to about 11.

7 . The method of claim 1 , wherein the concentration of clostripain is about 0.01 to about 3.0 units of clostripain per about 2 to about 5 mg polypeptide.

8 . The method of claim 1 , wherein the cleavage is performed in the presence of about 0.5 mM to about 10 mM CaCl 2 .

9 . The method of claim 3 , wherein the Linker comprises Pro-Gly-Xaa 1 -Xaa 2 ; and Xaa 1 is aspartic acid.

10 . The method of claim 3 , wherein the Linker comprises Val-Asp-Xaa 1 -Xaa 2 ; and Xaa 1 is aspartic.

11 . The method of claim 3 , wherein the Linker comprises Ile-Thr-Xaa 1 -Xaa 2 ; and Xaa 1 is aspartic acid.

12 . The method of claim 3 , wherein Peptide 5 is selected from the group consisting of Gly-Ser-, Cys-His-, Cys-His-Xaa-Xaa- and Val-Asp-; Xaa 1 is Aspartic acid or Glutamic acid; Xaa independently is any amino acid residue other than Arginine and m is an integer of 1 to 10.

13 . The method of claim 1 , wherein the desired peptide comprises anyone of SEQ ID NO:1-16.

14 . The method of claim 1 , wherein the polypeptide comprises a soluble four copy or six copy GLP-2(1-34) sequence.

15 - 71 . (canceled)

Assignments (1)
MERGER Recorded Dec 3, 2009
From: RESTORAGEN, INC.
To: MEDTRONIC, INC.
Reel/Frame 023594/0372 →