Preparation of biologically active recombinant human progastrin(1-80).
The bacterial expression of human progastrin(6-80) has been reported previously [Baldwin, G.S. et al. (2001) J. Biol. Chem. 276: 7791-7796]. The aims of the present study were to prepare full-length recombinant human progastrin(1-80) and to compare its biological activity with that of progastrin(6-80) in vitro, to determine whether or not the N-terminal five amino acids contributed to activity. A fusion protein of glutathione-S-transferase and human progastrin(1-80) was expressed in Escherichia coli, collected on glutathione-agarose beads, and cleaved with enterokinase. Progastrin(1-80) was purified by reversed-phase and anion exchange HPLC and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. No differences were detected in the extent of stimulation by progastrin(1-80) and progastrin(6-80) in proliferation and migration assays with the mouse gastric cell line IMGE-5. We conclude that residues 1-5 of progastrin(1-80) are not essential for biological activity.
McQueen
Kim
K
University of Melbourne Department of Surgery, Austin and Repatriation Medical Centre, Melbourne, Victoria, Australia.
Kovac
Suzana
S
Ho
Po-Ki
PK
Rorison
Kristy
K
Pannequin
Julie
J
Neumann
Greg
G
Shulkes
Arthur
A
Baldwin
Graham S
GS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005753:Gastric Mucosa / Q000166:cytology; D005755:Gastrins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization
D000596:Amino Acids; D005755:Gastrins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C013706:big gastrin; D005982:Glutathione Transferase