Cloning, recombinant expression and purification of turbot （Scophthalmus maximus） mature IGF-I

Insulin-like growth factor I （IGF-I） is a conserved peptide expressed ubiquitously, which shows highly homologous diverse effects on development, growth, and metabolism. With RT-PCR, the fragment encoding the turbot （Scophthalmus maximus） mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which may form 3 disulfide bonds. The target fragment was then successfully subcloned into the express vector pGEX-4T-1 and was highly expressed in E.coli BL21（DE3）plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 ku and maximally amounted to 59 % of the whole protein in the E.coli cell 4 hours after being induced with IPTG. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. The inclusion bodies were dissolved in 6 mol/Lguanidine chloride followed by pulse renaturation in refolding buffer containing 0.5 mol/L L-Arginine, 1.0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysed, and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells.