Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein.
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 alpha-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 alpha-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.
Chow
B K
BK
University of British Columbia, Department of Biochemistry, Vancouver, Canada.
Morrow
G W
GW
Ho
M
M
Pederson
R A
RA
McIntosh
C H
CH
Brown
J C
JC
MacGillivray
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D004926:Escherichia coli / Q000378:metabolism*; D005749:Gastric Inhibitory Polypeptide / Q000096:biosynthesis*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013205:Staphylococcal Protein A / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D005749:Gastric Inhibitory Polypeptide; D013917:Thrombin