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.
|Number of pages||7|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Nov 1993|
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