添加乙酸增强大肠杆菌中外源蛋白兔PKCepsilon催化功能域的溶解性表达

Enhancing Effect of Supplemental Acetate on Soluble Expression of Rabbit PKCepsilon Catalytic Domain in Escherichia Coli

目的研究在E.coli培养液添加葡萄糖增加外源蛋白的溶解性表达与其可能的机制.方法将兔蛋白激酶C(PKCepsilon)催化功能域(PKCeC)基因重组入表达载体pUC18中,在大肠杆菌中以与蛋白A融合的形式表达,向表达培养液中添加不同浓度的葡萄糖、乙酸、磷酸盐等和改变诱导时间,用SDS-PAGE和Westernblot检查PKCeC在细菌细胞浆和包涵体中的分布比例变化.结果细菌培养液中添加葡萄糖量增加,兔PKCeC表达时间延后,胞浆中其溶解性表达量增加,伴随葡萄糖消耗发生的培养液pH降低;直接添加乙酸增强PKCeC溶解性的表达,在此基础上添加磷酸盐可以抵消乙酸增强溶解性表达的效用.结论在培养液中添加葡萄糖可以有效地增加兔PKCeC蛋白在E.coli中的溶解性表达,其表达时间与添加葡萄糖量成正相关,并且葡萄糖的这种作用很可能与葡萄糖在细菌中代谢所伴随的乙酸生成所致的pH下降有关.

Objective To investigate the possible mechanisms of supplemental glucose in edhancirig soluble expression of extragenic protein in E. coli culture. Methods The coding region of catalytic domain of rabbit protein kinase C epsilon （PKCeC） was cloned into pUC18 vector and expressed as a fusion protein with IgG binding domain of staphylococcal protein A. The culture conditions, such as adding of vary concentrations of supplemental components （glucose, acetate and phosphate） and induction duration was invested for enhancing soluble expression of PKCeC. SDS-PAGE and western blot were used to check the expressed protein distribution between cytoplasm and inclusion bodies. Results Addition of glucose into bacterial culture was necessary for soluble expression of PKCeC. The expression of PKCeC was delayed while increasing the glucose concentration.Adding acetate into bacterial culture enhanced the soluble expression of PKCeC further. The effect of acetate on soluble expression of PKCeC was countervailed by addition of phosphate. Gonelusion Addition of glucose in bacterial culture is necessary for soluble expression of PKCeC in E. coli. Such effect of glucose may be based on the acetate production which accompanies the metabolisms of glucose in E. coli. In turn, the effect of acetate come from the acidic change of culture result from increase of acetate in culture.