Method of producing insulinotropic GLP-1 (7-36) polypeptide and/or GLP-1 analogs
The present invention discloses a method of producing polypeptides, including insulinotropic GLP-1 (7-36) polypeptide and/or GLP-1 analogs, by ligating genes in a tandem way. Also disclosed are the recombinant polypeptides produced by such a method. Using the method of this invention, 1 to 32 copies of GLP-1 (7-36) and/or GLP-1 analog genes may be expressed in tandem and the desired polypeptide can be obtained after cleavage of a fusion protein and further processes of separation and purification thus making possible the production of recombinant polypeptides, including recombinant GLP-1 (7-36) and/or GLP-1 analogs on a large scale, at a significantly reduced production cost.
1. 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.
2. The method according to claim 1 wherein the two restriction endonucleases capable of forming a hybrid site are Bgl II and BamH I.
3. The method according to claim 1 wherein the two restriction endonucleases capable of forming a hybrid site are Sal I and Xho I.
4. The method according to claim 1 in which said vector contains N copies of the GLP-1 (7-36) gene, GLP-1 analog gene, or a combination of GLP-1 (7-36) gene and GLP-1 analog gene, wherein N is 4.
5. The method according to claim 1 in which the said vector contains N copies of the GLP-1 (7-36) gene, GLP-1 analog gene, or a combination of GLP-1 (7-36) gene and GLP-1 analog gene, wherein N is an integer from 8 to 32.
6. The method according to claim 5 in which the said vector contains N copies of the GLP-1 (7-36) gene, GLP-1 analog gene, or a combination of GLP-1 (7-36) gene and GLP-1 analog gene, wherein N is 16.
7. The method according to claim 5 in which the said vector contains N copies of the GLP-1 (7-36) gene, GLP-1 analog gene, or a combination of GLP-1 (7-36) gene and GLP-1 analog gene, wherein N is 32.
8. The method according to claim 1 in which said host cell expresses a protein containing N copies of the GLP-1 (7-36) polypeptide, GLP-1 analog, or the combination thereof, wherein N is 4.
9. The method according to claim 8 wherein said host cell is a prokaryotic cell.
10. The method according to claim 9 wherein said host cell is Escherichia coli JM103, JM109, HB101, or DH5α or C600.
11. The method according to claim 1 in which said host cell expresses a protein containing N copies of the GLP-1 (7-36) polypeptide, GLP-1 analog, or the combination thereof, wherein N is an integer from 8 to 32.
12. The method according to claim 11 in which said host cell expresses a protein containing N copies of the GLP-1 (7-36) polypeptide, GLP-1 analog, or the combination thereof, wherein N is 16.
13. The method according to claim 11 in which said host cell expresses a protein containing N copies of the GLP-1 (7-36) polypeptide, GLP-1 analog, or the combination thereof, wherein N is 32.
14. The method according to claim 1 wherein said protein is cleaved at step (f) by clostripain or trypsin.
15. 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.