蓝杆藻ATCC51142中琥珀酸半醛脱氢酶的原核表达及生化表征  

Expression and characterization of succinic semialdehyde dehydrogenase from Cyanothece sp. ATCC51142

作　　者：谢琮琮 李至敏[2] 王小琴[1] 雷国风 张伟 李志敏[1,3] XIE Cong-Cong;LI Zhi-Min;WANG Xiao-Qin;LEI Guo-Feng;ZHANG Wei;LI Zhi-Min(College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang,Jiangxi 330045,China;College of Science,Jiangxi Agricultural University,Nanchang,Jiangxi 330045,China;Jiangxi Key Laboratory for Conservation and Utilization of Fungal Resources,Jiangxi Agricultural University,Nanchang,Jiangxi 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 [3]江西农业大学菌物资源保护与利用江西省重点实验室,江西南昌330045

出　　处：《微生物学通报》2019年第11期2933-2943,共11页Microbiology China

基　　金：国家自然科学基金(31560250,31860249);江西省自然科学基金重大项目(20161ACB21012);江西省教育厅科技研究重点项目(GJJ160353)~~

摘　　要：【背景】蓝藻中生成琥珀酸的三羧酸循环途径与其他物种不同。由于α-酮戊二酸脱羧酶和琥珀酸半醛脱氢酶的存在使得蓝藻的三羧酸循环途径变得完整。琥珀酸半醛脱氢酶催化琥珀酸半醛氧化为琥珀酸,在蓝藻中广泛存在。【目的】克隆、表达和纯化蓝杆藻ATCC51142中cce4228基因编码蛋白,并对其进行生化表征。【方法】以蓝杆藻ATCC51142基因组为模板克隆得到cce4228基因,将其插入到原核表达载体pET-28a上,在大肠杆菌BL21(DE3)细胞中进行异源表达,利用Ni-NTA树脂纯化cce4228蛋白。运用紫外分光光度法和生物信息学方法表征重组cce4228蛋白生化特性。【结果】构建了pET-28a-cce4228重组表达质粒,重组cce4228蛋白在大肠杆菌中得到可溶性表达,获得了纯度大于90%的cce4228蛋白。酶动力学测试和生物信息学分析结果显示,cce4228蛋白是一个NADP+-依赖型的琥珀酸半醛脱氢酶。【结论】蓝杆藻ATCC51142中cce4228基因编码一个偏好NADP+辅因子的琥珀酸半醛脱氢酶,cce4228蛋白的生化表征结果为进一步深入研究cce4228蛋白的结构功能关系及催化机制奠定了基础。[Background]The cyanobacterial tricarboxylic acid cycle,which produces succinic acid,is different from that of other species.The existences ofα-ketoglutarate decarboxylase and succinic semialdehyde dehydrogenase in cyanobacterium make its tricarboxylic acid cycle complete.Succinic semialdehyde dehydrogenases that catalyze the conversion of succinic semialdehyde to succinic acid,exist in cyanobacteria extensively.[Objective]Clone,express and purify the protein encoded by the cce4228 gene of Cyanothece sp.ATCC51142,and characterize its biochemical properties in vitro.[Methods]cce4228 gene was cloned into pET-28a vector,overexpression of recombinant cce4228 protein was induced by isopropylβ-D-thiogalactoside(IPTG)in E.coli BL21(DE3)and then the protein was purified by Ni-NTA affinity chromatography.The biochemical properties of cce4228 protein were characterized by spectrophotometric and bioinformatics methods.[Results]An expression plasmid pET-28a-cce4228 was constructed and cce4228 protein was overexpressed in soluble form in E.coli BL21(DE3).Recombinant cce4228 protein with purity higher than 90%was obtained.Steady-state kinetic and bioinformatic studies demonstrated that cce4228 protein was a NADP+-dependent succinic semialdehyde dehydrogenase.[Conclusion]The cce4228 gene in Cyanothece sp.ATCC51142 encodes a succinic semialdehyde dehydrogenase,which prefers to use NADP+rather than NAD+as its cofactor.These results will provide basis to understand the structure-function relationship and catalytic mechanism of cce4228 protein further.

关 键 词：蓝杆藻ATCC51142 琥珀酸半醛脱氢酶 原核表达 蛋白纯化及表征 

分 类 号：R73[医药卫生—肿瘤]