Prokaryotic expression of an active endogenous inhibitor of angiogenesis-canstatin / 中华消化杂志

ABSTRACT

Objective To express the recombinant human canstatin protein, and to examine its biological activity. Methods Canstatin cDNA was cut off from the plasmid pUCm-T/canstatin with restriction enzymes BamHⅠ and Hind Ⅲ. The cDNA fragment was then ligated into the correspondence sites of plasmid pET-22b(+) by T4 DNA ligation enzyme and transformed into E.coli BL21 which was induced to express proteins with isopropyl-1-thio-b-dgalactopyranoside (IPTG). The expressed proteins were analyzed by SDS-PAGE and purified through Ni-NTA column affinity chromatography. Chick chorioallantoic membrane (CAM) assay was performed to determine the activity of the recombinant protein. Results Canstatin cDNA from pUCm-T showed one clear objective DNA band with electrophoresis. Seven of positive colonies were selected and identified by restriction enzyme analysis with BamH Ⅰ and Hind Ⅲ. Electrophoresis revealed that all selected colonies had two specific bands,one near the location of primary plamid,the other near that of objective gene fragment. After IPTG induction, there was a new protein band about 24 000 on SDS-PAGE.The induced product over total bacterial proteins in 1,2, 3. and 4 hours after induction was 18. 2%, 18. 8%, 23.0% and 23.4%, respectively, by densitometry examination. CAM assay demonstrated that the recombinant canstatin protein significantly inhibited the embryonic neovascularization in a dose-dependent manner. Conclusion The prokaryotic expression vector of human canstatin gene has been successfully constructed, laying the foundation for further clinical study.