重组人胶原蛋白肽在大肠杆菌中的高效表达

目的:构建含有胶原蛋白基因的原核表达载体pET32a-CP6,并转入大肠杆菌BL21 (DE3)中进行高效表达,为获得大量可溶的胶原蛋白肽提供可靠依据.方法:将重组表达载体pET32a-CP6转化到大肠杆菌BL21中,以IPTG为诱导剂,对温度、接种量、诱导时机、IPTG诱导浓度等各种发酵参数进行优化,筛选高效表达的发...

Expression of recombinant human collagen peptide in E.coli

Objective To construct the recombinant expression vector pET32a-CP6 and transform into the Escherichia colic(E.coli) BL21(DE3) and express the recombinant protein at a high level,in order to provide reliable basis for obtaining a large number of soluble collagen.Methods The recombinant plasmid pET32a-CP6 was transformed into E.coli BL21 and induced by IPTG.The expression conditions of the recombinant protein were optimized in this test,which included temperature,time for induction,concentration of ITPG,inoculation volume,adding time of IPTG.The recombinant E.coli was fermented under the optimal condition,and the lysate of bacteria was purified by nickel ion affinity.The purified target protein was determined for purity by SDS-PAGE.Results The optimal temperature and time for induction of recombinant E.coli were 37℃ and 5 h respectively,while the optimal concentration of IPTG as an inducer was 0.5 mmol·L-1.The expression level of target protein reached 31.52 mg·L-1 under the optimal condition.The Western blotting results showed that the recombinant protein had specific reaction with anti-human COL6A2 antibody.Conclusion The optimized recombinant E.coli can efficiently express the recombinant human collagen,the recombinant protein is purified successfully.