Cloning of human canstatin gene, expression and purification of its recombinant protein / 解放军医学杂志

ABSTRACT

Objective To clone human canstatin gene, construct its prokaryotic expression vector, express and purify its recombinant protein. Methods The total RNA was extracted from human placenta tissues. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting product was cloned into pUCm-T vector and sequenced. Then the confirmed canstatin cDNA was cloned into plasmid pET-22b(+) and then transformed into E.coli BL21 where it was induced to express proteins by isopropyl-1-thio-b-Dgalactopyranoside (IPTG). The expression of protein was analyzed through SDS-PAGE. Cells after induced 3 hours by IPTG were harvested, sonicated briefly and the proteins were purified through affinity chromatography. Results (1)The extracted total RNA was separated into three clear bands indicating 28S, 18S, and 5S after electrophoresis and the concentration was 1.8 g/L. (2)The target sequences were specifically amplified through RT-PCR. (3)The purified RT-PCR product was cloned into pUCm-T vector and then sequenced, demonstrating to be the same as that of canstatin gene in GenBank. (4) the expression vector pET-22b(+) was constructed and verified by the method of BamH Ⅰ and Hind Ⅲ digestion. (5) After IPTG induction, there was a new protein band about Mr 24kD on SDS-PAGE. The percent expressed product over total bacterial proteins after 1, 2, 3, and 4 hours of induction was 18.2%, 18.8%, 23.0% and 23.4%, respectively, estimated by densitometry. (6)After affinity chromatography, SDS-PAGE showed only one clear band existed in 125 mmol/L or 250 mmol/L imidizone elution. Conclusion Human canstatin gene has been successfully cloned and its prokaryotic expression vector has also been successfully constructed. Further more, purified recombinant proteins are obtained through affinity chromatography, laying foundation for further study of its clinical application.