中国生物工程杂志

Human (homo sapiens) insulin-like growth factor-1 (hIGF-1) gene coding for the mature protein was synthesized and cloned in a pMD18-T vector, and then transferred into prokaryotic fusion expression vector pGEX-2T. This fused hIGF-1-GST gene was under the control of tac promoter and the fusion expression of hIGF-1 with an Glutathione S-transferase (GST) tag in the E. coli BL21(DE3) upon induction with isopropyl-1-thio-beta-D-galactoside (IPTG). The recombinant protein showed a molecular weight of about 35 kD and was mainly in the form of inclusion bodies according to SDS-PAGE. The insoluble hIGF-1-GST was solublized from inclusion bodies by denaturation using 6 M urea in the presence of DTT. Both the recombinant hIGF-1-GST from the soluble fraction and the dissolved inclusion bodies of the crude extract of culture could be purified to over 90% purity by using GST-tag affinity chromatography. The purified hIGF-1-GST was again confirmed by SDS-PAGE and Western blot analysis. The expressed protein contained within the inclusion bodies was refolded and renatured to natural structure. After cleavage of the soluble and renatured fusion protein with thrombin, the released hIGF-1 was respectively purified by GST-tag affinity chromatography again and prepared for MTT colorimetric assays. Both the two hIGF-1 preparations could promote NIH/3T3 cells proliferation in a dose-dependent manner. The bioactivity of the soluble hIGF-1 was as potent as that of the standard hIGF-1 in cell culture bioassay