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 cleaving a peptide bond of a polypeptide, comprising:
combining the polypeptide with clostripain;
wherein the polypeptide is any amino acid sequence containing within it at least a fragment having an amino acid sequence of Formula I
-Xaa 1 -Xaa 2 -Xaa 3 - (I);
wherein Xaa 1 is a residue of aspartic acid, glycine, proline or glutamic acid;
Xaa 2 is an arginine residue;
Xaa 3 is any amino acid residue other than an acidic amino acid residue;
wherein the cleavage is performed between a pH of 6 to 7.0 and at a temperature ranging from 20° C. to 30° C.;
wherein the peptide bond cleavage occurs between Xaa 2 and Xaa 3 ; and
wherein the cleavage between Xaa 2 and Xaa 3 of Formula I occurs selectively over the cleavage of 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 concentration of clostripain is about 0.01 to about 3.0 units of clostripain per about 2 to about 5 mg polypeptide.
4. The method of claim 1 wherein the cleavage is performed in the presence of about 0.5 mM to about 10 mM CaCl 2 .
5. The method of claim 1 wherein the desired peptide comprises any one of SEQ ID NO:1-16.
6. The method of claim 2 wherein the polypeptide comprises four or six copies of GLP-2(1-34) sequence.