Recombinant coho salmon insulin-like growth factor I

Expression in Escherichia coli, purification and characterization

Authors


  • Note. The novel amino sequence data published here have been deposited with the EMBL SWISS-PROT protein sequence data banks and are available under accession number P17085.

Correspondence to S. Moriyama, c/o E. M. Plisetskaya, School of Fisheries HF-15, University of Washington, Seattle, Washington 98195, USA
Fax:+1 206 685 3275.

Abstract

Recombinant coho salmon insulin-like growth factor I (rsIGF-I) was produced in Escherichia coli, purified and characterized. The rsIGF-I expression vector was constructed by polymerase chain reaction and cloning into a plasmid containing a phage T7 RNA polymerase promoter. The rsIGF-I was recovered from bacterial inclusion bodies, solubilized under reducing conditions, immediately refolded, then fractionated by a two-step ion-exchange chromatography on DEAE-52 and Mono-S columns. It was further purified by HPLC on a reverse-phase Asahi-Pak C4P-50 C4 column. Purification of rsIGF-I was monitored by SDS/PAGE and immunoblot with anti-[human somatomedin C (SM C)/IGF-I] serum. The rsIGF-I appeared as a single band with molecular mass of 7 kDa, the same size as recombinant human IGF-I (rhIGF-I) and cross-reacted with anti-(human SM C/IGF-I) serum. The amino acid sequence of rsIGF-I contained an NH2-terminal methionine residue followed by the sequence predicted for mature sIGF-I. At concentrations in the range 3.9–250 ng/ml, rsIGF-I significantly stimulated sulfate uptake by the cultured branchial cartilage of coho salmon. The stimulatory effect of rsIGF-I was concentration dependent and slightly more potent than that of rhIGF-I at the highest concentration tested.

Abbreviations
GH

growth hormone

IGF-I

insulin-like growth factor I

IGF-II

insulin-like growth factor II

PCR

polymerase chain reaction

rcIGF-I

recombinant chicken insulin-like growth factor I

rhIGF-I

recombinant human insulin-like growth factor I

rsIGF-I

recombinant coho salmon insulin-like growth factor I

SM C

somatomedin C

Ancillary