METHOD FOR UNIVERSAL ENZYMATIC PRODUCTION OF BIOACTIVE PEPTIDES

التفاصيل البيبلوغرافية
العنوان: METHOD FOR UNIVERSAL ENZYMATIC PRODUCTION OF BIOACTIVE PEPTIDES
Document Number: 20120156719
تاريخ النشر: June 21, 2012
Appl. No: 12/354053
Application Filed: January 15, 2009
مستخلص: 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.
Inventors: Wagner, Fred W. (Walton, NE, US); Luan, Peng (Omaha, NE, US); Xia, Yuannan (Lincoln, NE, US); Strydom, Daniel (Lincoln, NE, US); Merrifield, Edwin H. (Lynnwood, WA, US); Bossard, Mary J. (Madison, AL, US); Holmquist, Barton (Eagle, NE, US); Seo, Jin Seog (Brampton, CA)
Claim: 1-2. (canceled)
Claim: 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-Xaa3-Peptide1)n-Linker-Xaa3-Peptide1  (III); wherein the desired peptide comprises Xaa3-Peptide1; Peptide1 is any amino acid sequence other than Xaa1-Xaa2; n is an integer ranging from 0 to 50; Xaa3 is not an acidic amino acid; Linker is a cleavable peptide linker having Formula (IV): (Peptide5)m—Xaa1-Xaa2  (IV); wherein m is an integer ranging from 0 to 50; Xaa1 is aspartic acid, glycine, proline or glutamic acid; Xaa2 is arginine; and Peptide5 is any single amino acid residue or amino acid sequence not containing Xaa1-Xaa2.
Claim: 4. The method of claim 1, wherein the polypeptide is a soluble polypeptide.
Claim: 5. The method of claim 1, wherein the cleavage is performed at about 18° C. to about 25° C.
Claim: 6. The method of claim 1, wherein the cleavage is performed between a pH of about 5 to about 11.
Claim: 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.
Claim: 8. The method of claim 1, wherein the cleavage is performed in the presence of about 0.5 mM to about 10 mM CaCl2.
Claim: 9. The method of claim 3, wherein the Linker comprises Pro-Gly-Xaa1-Xaa2; and Xaa1 is aspartic acid.
Claim: 10. The method of claim 3, wherein the Linker comprises Val-Asp-Xaa1-Xaa2; and Xaa1 is aspartic.
Claim: 11. The method of claim 3, wherein the Linker comprises Ile-Thr-Xaa1-Xaa2; and Xaa1 is aspartic acid.
Claim: 12. The method of claim 3, wherein Peptide5 is selected from the group consisting of Gly-Ser-, Cys-His-, Cys-His-Xaa-Xaa- and Val-Asp-; Xaa1 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.
Claim: 13. The method of claim 1, wherein the desired peptide comprises anyone of SEQ ID NO:1-16.
Claim: 14. The method of claim 1, wherein the polypeptide comprises a soluble four copy or six copy GLP-2(1-34) sequence.
Claim: 15-71. (canceled)
Current U.S. Class: 435/681
Current International Class: 12
رقم الانضمام: edspap.20120156719
قاعدة البيانات: USPTO Patent Applications