Patent ID: 7544512

Claim:
A method of producing insulinotropic GLP-1 (7-36) polypeptide and/or GLP-1 analogs comprising: (a) introducing into a DNA fragment first and second individual restriction endonuclease cleavage sites capable of forming a hybrid site to two terminals of a gene which encodes either the GLP-1 (7-36) polypeptide or GLP-1 analogs, respectively, and a third restriction endonuclease cleavage site located outside of the region between said first and second restriction endonuclease cleavage sites, wherein the second restriction endonuclease cleavage site is located between the first and third restriction endonuclease cleavage sites; (b) digesting the DNA fragment of step (a) with restriction endonucleases at the first and third restriction endonuclease cleavage sites and ligating said digested DNA fragment with a vector, wherein said vector has the second and third restriction endonuclease cleavage sites and is digested at said second and third restriction endonuclease cleavage sites before said ligation; (c) repeating step (b) to form an expression vector comprising N copies of GLP-1 (7-36) gene, GLP-1 analog gene, or a combination of GLP-1 (7-36) gene and GLP-1 analog genes, wherein N is an integer from 2 to 32; (d) transforming said expression vector into a host cell; (e) expressing in the host cell a protein comprising N copies of the GLP-1 (7-36) polypeptide, GLP-1 analog, or the combination thereof, but without any carrier protein; (f) breaking up the host cell and cleaving said protein of step (e) under conditions wherein the cleavage does not occur at the peptide bonds adjacent to internal lysine residues to obtain GLP-1 (7-36) polypeptides or GLP-1 analogs that are capable of stimulating the secretion of insulin; and (g) separating and purifying said GLP-1 (7-36) polypeptides or GLP-1 analogs.