布氏锥虫亮氨酰-tRNA合成酶的克隆表达纯化及活性测定  

Cloning,expression,purification and activity assay of Leucyl-tRNA synthetase from Trypanosoma Brucei

作　　者：姚璎[1] 杲光伟[1] 李大伟[1] 

机构地区：[1]上海交通大学药学院,上海200240

出　　处：《中国生化药物杂志》2010年第1期14-18,共5页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的克隆布氏锥虫亮氨酸-tRNA合成酶基因,并进行表达纯化及活性测定。方法通过PCR方法从布氏锥虫细胞基因组中扩增出亮氨酰-tRNA合成酶(LeuRS)基因,依次克隆入pBS-T克隆载体及pET21a(+)表达载体,在大肠杆菌BL21(DE3)RIPL中通过条件优化后表达。表达产物用His-Bind亲和色谱纯化,并用免疫印迹进行鉴定。采用放射性同位素方法进行酶活性测定。结果PCR扩增得到3.2 kb的DNA片段,重组质粒pET21a(+)/LeuRS经酶切鉴定和测序分析,表明构建正确。表达的重组蛋白占菌体可溶性蛋白的20%,纯化后蛋白纯度达85%,免疫印迹进行鉴定蛋白正确,酶活性测定计算出提纯物酶活性约为72 U/mL。结论已成功克隆表达纯化出布氏锥虫亮氨酸-tRNA合成酶,并用放射性同位素方法进行了酶活性测定,这为下一步进行布氏锥虫亮氨酰-tRNA合成酶抑制物的设计和体外筛选奠定了良好的基础。Purpose To clone and express Trypanosoma Leucyl-tRNA synthetase （LeuRS） gene and to complete the purification and activity assay of LeuRS. Methods We cloned the LeuRS gene from T. Brucei ge- nomic DNA,and cloned it into pBS-T and then into the expression vector pET21a（ ＋ ）. The expression of T. Brucei LeuRS was carried out in E. coli BL21 （DE3）RIPL host by optimizing the expression conditions. The products are purified by His-Bind affinity column and verified by Western blot . Enzymatic activity was detected by isotope labeling. Results A DNA fragment at length of 3.2 kb was amplified. Both restriction analysis and sequencing analysis proved that recombinant plasmid pET21a（ ＋ ）/LeuRS was correctly constructed. The ex- pressed product contained about 20 % of total somatic soluble protein and reached a purity of 85 % after purifi- cation. It was verified by the Western blot. Enzyme unit per millilitre of purified products was about 72. Con- elusiou Here we report the successful clone, expression and purification of LeuRS from Trypanosoma Brucei （ T. Brucei） in bacterial host. In addition, we tested its enzymatic activity by isotope labeling. This work would be helpful to the design and in vitro screening of Trypanosoma LeuRS inhibitors.

关 键 词：布氏锥虫 亮氨酰-TRNA合成酶 原核表达 纯化 酶活测定 

分 类 号：Q78[生物学—分子生物学]