Expression and purification of human cardiotrophin-1 in E.coli and identification of its biological activity / 第三军医大学学报

ABSTRACT

Objective To express recombinant human cardiotrophin 1 (huCT 1) protein with biological activity in E coli Methods huCT 1 open reading frame gene from plasmid pBSSK huCT1 was cloned into plasmid pMD18 T by PCR and T A cloning, and then cloned into prokaryotic GST fusion expression vector pGEX 2T to give pGEX2T huCT1 After pGEX2T huCT1 expression was induced by IPTG in E coli at different temperature and different time, the expression level and the proportion of the soluble protein were analyzed by SDA PAGE Then the soluble GST/huCT1 was purified by immobilized glutathione columns The GST fusion protein was cleaved by thrombin and purified again The recombinant huCT 1 with biological activity was identified according to the survival of axotomized sciatic motoneurons Results After the induction of pGEX2T huCT1 DH5? cells by IPTG at 29 ℃ for 4 h, the highest expression level of the recombinant protein was about 1/5 of total cell proteins, and the soluble portion was about 2/5 of fusion protein Purification of the soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST fusion protein and huCT 1 protein, respectively Recombinant huCT 1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats Conclusion Recombinant huCT 1 has biological activity in rat neurons