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Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein |
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| Authors: | Billy K. -C. Chow Glenn W. Morrow Margaret Ho Raymond A. Pederson Christopher H. S. McIntosh John C. Brown Ross T. A. MacGillivray |
| Affiliation: | a University of British Columbia, Department of Biochemistry, Vancouver, B. C., Canada V6T 1W5 b University of British Columbia, The MRC Regulatory Peptide Group, Department of Physiology, Vancouver, B. C., Canada V6T 1W5 |
| Abstract: | Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas. |
| Keywords: | Human GIP Expression in E. coli Protein A fusion protein Thrombin cleavage |
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