Optimization of expression conditions and activity identification of hepatocyte-targeting peptide-human endostatin / 生物工程学报

ABSTRACT

To obtain sufficient purified and active fusion protein-hepatocyte-targeting peptide-human endostatin (HTP-rES), we studied the growth curve and the optimal induction timing of BL21/pET21b-HTP-rES. Different conditions of pH value, induction time, induction concentration and induction temperature were optimized by univariate analysis. After washing, refolding and purifying, the activity of fusion protein was identified by flow cytometry and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT). Results show that the logarithmic growth phase of BL21/pET21b-HTP-rES was from 1.5 h to 3.5 h, the optimum expression conditions were pH 8.0, 0.06 mmol/L IPTG, at 42 ℃ for 5 h. The purity of inclusion bodies was up to 60% after washing. The purity of target protein was more than 95% after refolding and purification. Our findings provide the foundation for further biological activity and drug development.