Characterization of Two Forms of Recombinant Salmon Insulin-Like Growth Factor-I: Activities and Complexing with Insulin-Like Growth Factor-I Binding Proteins

Abstract

Coho salmon insulin-like growth factor I (IGF-I) with N-terminal glycine residue ([Gly]-rsIGF-I) was derived from methionine-extended recombinant salmon IGF-I ([Met]-rsIGF-I) produced in Escherichia coli according to [18]. Purified [Met]-rsIGF-I was treated with methionine aminopeptidase (MAP) to remove the N-terminal Met residue and resultant [Gly]-rsIGF-I was further purified by HPLC on a reverse-phase C4 column. The partial N-terminal amino acid sequence (residues 1–25) of [Gly]-rsIGF-I was identical to that of the native salmon IGF-I. [Gly]-rsIGF-I appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a single band with a molecular weight of 7 kDa and cross-reacted with an antibody against [Met]-rsIGF-I in immunoblot and radioimmunoassay (RIA). In the salmon IGF-I RIA, the dilution curves of [Gly]-rsIGF-I were parallel to the [Met]-rsIGF-I standard curves and the two peptides appeared to be equally potent in displacing label. At concentrations 50 and 100 ng/ml, [Gly]rsIGF-I significantly stimulated the sulfate uptake by the cultured salmon branchial cartilage in a dose-dependent manner. The stimulatory effect of [Gly]-rsIGF-I was equipment to that of [Met]-rsIGF-I. Used as probes in a Western ligand blotting, both [Gly]-rsIGF-I and [Met]-rsIGF-I were able to bind to two IGF binding proteins (IGFBPs) of 23 kDa and 28 kDa from salmon hepatocyte culture medium. The binding could be prevented by preincubation with either unlabeled [Gly]-or [Met]-rsIGF-I. These results indicate that [Gly]-rsIGF-I, which is identical to the native form of IGF-I, and the Met-extended form of recombinant salmon IGF-I, are equally biologically and immunologically active and may substitute for each other in physiological experiments.