表达大肠杆菌谷氨酸-1-半醛氨基转移酶对红色荧光报告蛋白尿卟啉原Ⅲ甲基化酶的影响

Effect of the expression of Escherchia coli glutamate-1-semiadhyde aminotransferase on the red fluorescent protein uroporphyrinogen Ⅲ methyltransferase

谷氨酸-1-半醛氨基转移酶(Glutamate-1-semiadhyde aminotransferase,GSAT)是尿卟啉原Ⅲ生物合成上游途径的一个酶,尿卟啉原Ⅲ是红色荧光报告蛋白尿卟啉原Ⅲ甲基化酶(Uroporphyrinogen Ⅲ methyltransferase,UPMT)的底物。为了探明大肠杆菌共表达GSAT对UPMT荧光强度的影响,通过PCR扩增玉米upmt基因,将其插入pETDuet-1质粒中第2个顺反子中,构建的载体命名为pETU,表达UPMT的N端含有组氨酸标签;通过PCR扩增大肠杆菌编码GSAT的hemL基因,定点突变去除hemL基因中NcoⅠ序列,亚克隆至pET-51b质粒,再将获得的hemL基因插入pETU质粒的第一个顺反子中,构建pETeGU载体。表达GSAT的N端含有Strep标签。和单独表达upmt基因相比,表达2个基因后,蛋白印迹分析表明没有明显改变UPMT表达量,光谱扫描分析显示没有改变荧光物质的组成,但是增强了重组细胞的红色荧光物质三甲基咕啉的含量,该物质在354nm有特异吸收。用2mmol/L的GSAT抑制剂3-氨基-2,3二羟基苯甲酸处理后,表达两种酶的菌落荧光消失,表明重组GSAT可能增加内源尿卟啉原Ⅲ水平,从而增强重组UPMT催化产生的红色荧光。

Glutamate-1-semiadhyde aminotransferase（GSAT） is an enzyme in the upstream biosynthetic pathway of uroporphyrinogen III that is the substrate of uroporphyrinogen III methyltransferase（UPMT）,a novel red fluorescent protein.In order to detect the effect of overexpression of GSAT with UPMT on the fluorescent intensity in Escherichia coli,we amplified maize upmt gene by PCR and inserted into the first cistron of pET Duet-1 plasmid to create the vector pETU.The expressed UPMT was fused histidine tag at N terminus.We also amplified E.coli hemL gene encoding GSAT by PCR reaction,eliminated Nco I site within the hemL gene by site-directed mutagenesis and subcloned into pET-51b plasmid.The resultant hemL gene was inserted the second cistron of pETU plasmid to produce the vector pETeGU.The expressed GSAT has the extra Strep·TagII at N terminus.Compared to overexpression upmt gene alone,coexpression both genes did not resulted in the remarkable change in either the amount of the UPMT,as estimated by western blot analysis,or the constitution of red fluorescent materials,as shown by UV/visible light scanning analysis,but increased cellular level of the fluorescent material trimethylpyrrocorphin with the specific absorption at 354 nm.The red fluorescence emitted by the colonies cooverexpressing both enzymes completely disappeared after treated by 2 mmol/L gabaculine,the GSAT inhibitor,suggested that the recombinant GSAT may increase the cellular level of uroporphyrinogen Ⅲ,and thus enhanced the red fluorescence of the E.coli cells conferred by the recombinant UPMT.