Method for universal enzymatic production of bioactive peptides
View Patent ↗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.
1. A method for producing a desired peptide from a polypeptide, comprising:
combining the polypeptide and clostripain, wherein
the polypeptide comprises Formula (II)
(Xaa 3 -Peptide 1 -Xaa 1 -Xaa 2 ) n -Xaa 3 -Peptide 1 -Xaa 1 -Xaa 2 (II);
the desired peptide comprises Xaa 3 -Peptide 1 -Xaa 1 -Xaa 2 ;
Peptide 1 is any amino acid sequence other than Xaa 1 -Xaa 2 ;
n is an integer ranging from 0 to 50;
Xaa 1 is aspartic acid, glycine, proline or glutamic acid;
Xaa 2 is arginine; and
Xaa 3 is not an acidic amino acid;
wherein the peptide bond between Xaa 2 and the amino acid on the C-terminal side of Xaa 2 is cleaved; and
wherein the cleavage occurs selectively over cleavage at other arginine or lysine residues within the polypeptide.
2. The method of claim 1 , wherein the polypeptide is a soluble polypeptide.
3. The method of claim 1 , wherein the cleavage is performed at about 18° C. to about 25° C.
4. The method of claim 1 , wherein the cleavage is performed between a pH of about 5 and about 11.
5. 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.
6. The method of claim 1 , wherein the cleavage is performed in the presence of about 0.5 mM to about 10 mM CaCl 2 .
7. The method of claim 1 , wherein the desired peptide comprises any one of SEQ ID NO:1-16.
8. The method of claim 1 , wherein the polypeptide comprises four or six copies of GLP-2(1-34) sequence.
9. The method of claim 1 , wherein the polypeptide comprises four or six copies of GLP-1(7-36) sequence.