Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Peptides. 1990 Nov-Dec;11(6):1069-74. doi: 10.1016/0196-9781(90)90132-o.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Biological Assay
  • Escherichia coli / metabolism*
  • Gastric Inhibitory Polypeptide / biosynthesis*
  • In Vitro Techniques
  • Molecular Sequence Data
  • Pancreas / chemistry
  • Perfusion
  • Rats
  • Rats, Inbred Strains
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / biosynthesis
  • Staphylococcal Protein A / metabolism*
  • Substrate Specificity
  • Thrombin

Substances

  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Staphylococcal Protein A
  • Gastric Inhibitory Polypeptide
  • Thrombin