集胞藻PCC6803中α-酮戊二酸脱羧酶的原核表达分析  

Prokaryotic Expression Analysis ofα-ketoglutaric Acid Decarboxylase from Synechocystis sp.PCC6803

作　　者：张伟 刘志文[1] 王小琴[1] 李至敏[2] 谢琮琮 李志敏[1] ZHANG Wei;LIU Zhi-wen;WANG Xiao-qin;LI Zhi-min;XIE Cong-cong;LI Zhi-min(College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China;College of Science,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学生物科学与工程学院,江西南昌330045 [2]江西农业大学理学院,江西南昌330045

出　　处：《江西农业大学学报》2020年第3期597-607,共11页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31860249);江西省自然科学基金项目(20161ACB21012,20192BAB204011)。

摘　　要：【目的】α-酮戊二酸脱羧酶(α-ketoglutaric acid decarboxylase,α-KGD)是蓝细菌三羧酸循环途径中的一个关键酶。试验旨在优化集胞藻PCC6803中sll1981基因编码蛋白的原核表达方法,并对不同表达方法获得的重组sll1981蛋白进行活性测定。【方法】利用分子生物学技术构建含有sll1981基因的多种表达载体,通过SDSPAGE分析这些表达载体在大肠杆菌表达系统中的表达和可溶性情况,利用Ni-NTA亲和层析分离纯化重组sll1981蛋白,然后通过酶偶联法测定重组sll1981蛋白的催化活性。【结果】含有sll1981基因的不同表达载体在大肠杆菌表达系统中的表达良好,然而重组sll1981蛋白的可溶性和催化活性差异较大。【结论】当pET28bsll1981质粒与pTf16分子伴侣质粒在大肠杆菌中共表达时重组sll1981蛋白的可溶性和催化活性均为最佳。酶动力学测试表明集胞藻PCC6803中sll1981基因编码蛋白具有α-酮戊二酸脱羧酶功能。为进一步研究α-酮戊二酸脱羧酶的结构功能关系及催化机制提供重要基础。[Objective]α-Ketoglutaric acid decarboxylase(α-KGD)was a key enzyme in the cyanobacterial tricarboxylic acid cycle.The aim of this study was to optimize the prokaryotic expression of protein encoded by sll1981 gene from Synechocystis sp.PCC6803 and to determine the catalytic activity of recombinant sll1981 protein obtained by different expression methods.[Method]Multiple expression vectors containing sll1981 gene were constructed by using molecular biology techniques.The expression and solubility of recombinant sll1981 protein in Escherichia coli were analyzed by SDS-PAGE.The recombinant sll1981 protein was isolated and purified by Ni-NTA affinity chromatography.The catalytic activity of the recombinant sll1981 protein was determined by enzymatic coupling method.[Result]It was found that different expression vectors containing sll1981 gene were well expressed in the expression system of Escherichia coli,whereas the solubility and catalytic activities of recombinant sll1981 protein were considerably different.[Conclusion]Purified recombinant sll1981 protein displayed optimal solubility and catalytic activity when pET28b-sll1981 plasmid and pTf16 chaperone plasmid were co-expressed in Escherichia coli.Enzymatic kinetics showed that the recombinant sll1981 protein was anα-ketoglutarate decarboxylase.This study provides an important basis for further research on the structure and function relationship and catalytic mechanism ofα-ketoglutarate decarboxylase.

关 键 词：集胞藻PCC6803 α-酮戊二酸脱羧酶 原核表达 sll1981基因 分子伴侣 

分 类 号：Q786[生物学—分子生物学]