溶藻弧菌PEPCK蛋白原核表达及其乙酰化、琥珀酰化修饰的鉴定

Prokaryotic Expression of the PEPCK Protein of Vibrio alginolyticus and Identification of Its Acetylation and Succinylation

构建溶藻弧菌HY9901 PEPCK蛋白的原核表达载体、优化其表达条件,并鉴定该蛋白的乙酰化及琥珀酰化修饰。采用PCR克隆pepck基因,构建pET-28a-pepck并将其转入大肠杆菌BL21(DE3),对重组菌株的培养温度、IPTG浓度及时间进行优化,采用SDS-PAGE和Western blot分析蛋白的表达及乙酰化、琥珀酰化修饰。结果显示,pepck基因全长1 629 bp,重组菌株可以正确表达分子质量约60.9 kD的蛋白。诱导温度:在28℃条件下PEPCK存在于上清和包涵体中,在37℃则主要以包涵体形式存在;IPTG浓度:0.2 mmol/L-1.0 mmol/L范围内,PEPCK的表达量无明显差异;时间:在0-8 h内PEPCK表达量随时间增长逐渐增加,诱导8 h后趋于稳定。Western blot显示PEPCK蛋白具有乙酰化修饰和琥珀酰化修饰。成功构建溶藻弧菌PEPCK重组表达菌株,其最优诱导表达条件为0.2 mmol/L IPTG,28℃诱导8 h;经鉴定PEPCK蛋白具有乙酰化修饰和琥珀酰化修饰。

The main objective of this study is to construct a prokaryotic expression vector of PEPCK protein in Vibrio alginolyticus HY9901,to optimize its expression conditions and to identify its acetylation and succinylation.Firstly,the target gene pepck was cloned via PCR.An expression vector pET-28a-pepck was then constructed and transferred into Escherichia coli BL21(DE3).Subsequently,the culture temperature,IPTG concentration and time of the recombinant strain were optimized.In the end,the protein expression,acetylation and succinylation were analyzed by SDS-PAGE and Western blot.The final results of this study showed that the length of the pepck gene of V.alginolyticus strain HY9901 was 1629 bp,and its His-PEPCK fusion protein with 60.9 kD was successfully expressed in the recombinant strain.The PEPCK protein was distributed in the supernatant and inclusion body at 28℃,but existed mainly as inclusion body when the temperature was at 37℃.There was no significant difference in PEPCK expression while the IPTG concentration was in the range of 0.2-1.0 mmol/L.The PEPCK protein expression increased gradually with time and tended to be stable after 8 h when the induction time was within 0-8 h.Western blot results showed that the PEPCK protein was acetylated and succinylated.In conclusion,the recombinant expression strain for V.alginolyticus PEPCK is successfully constructed,and the recombinant protein is highly expressed under induction conditions with 0.2 mmol/L IPTG at 28℃for 8 h.The PEPCK protein is identified to be acetylated and succinylated.