重组人α降钙素基因相关肽在大肠杆菌中表达条件的优化

为提高人α降钙素基因相关肽(bαCGRP)在大肠杆菌中的表达量,研究了本室构建的pET-hαCGRP重组质粒在E.coli BL21trxB(DE3)pKysS宿主菌中的表达条件.经SDS-PAGE分析和YLN2000凝胶影像分析结果表明,重组菌以LB为培养基,氨苄青霉素浓度为50 mg/L,开始诱导时的菌体密度为OD600=0.6～0.8,所加IPTG的浓度为0.75mmol/L,37℃摇床180±5r/min振荡诱导培养4 h时,可获得高效表达的hαCGRP融合蛋白.重组蛋白的最高表达量占菌体总蛋白的70%～80%;它主要以可溶性形式存在,为下一步纯化工作提供了方便.

Optimization of Recombinant Human α Calcitonin Gene-related Peptide Expression in E. Coli

In order to improve the expression level of human α calcitonin gene-related peptide (hαCGRP), the growth conditions of the engineering strain, that confirmed by transforming the recombinant plasmid pET-hαCGRP which constructed by our team into the host E. coli BL21trxB(DE3)pLysS were studied, which remarkably influenced the final yield of protein expression. With the aid of SDS-PAGE and YLN2000 gel image analysis, it was found that the best expressed condition was that the induction was started as OD600 reached 0.6 ～ 0.8 by adding IPTG to a final concentration of 0.75 mmol/L and then continued incubation 4 hours at 37 ℃ (180 + 5 r/min) in the LB medium containing 50 mg/L ampicillin with shaking flasks. The maximum yield of fusion protein was about 70％ ～ 80％ of the total mass of bacterial proteins. Furthermore the soluble form of the target protein makes convenience for next purification work.