Cloning, expression, purification and activity assay of Leucyl-tRNA synthetase from Trypanosoma Brucei
- VernacularTitle:布氏锥虫亮氨酰-tRNA合成酶的克隆表达纯化及活性测定
- Author:
Ying YAO
;
Guangwei GAO
;
Dawei LI
- Publication Type:Journal Article
- Keywords:
Trypanosoma Brucei;
Leucyl-tRNA synthetase;
prokaryotic expression;
purification;
enzyme activity assay
- From:
Chinese Journal of Biochemical Pharmaceutics
2010;31(1):14-18
- CountryChina
- Language:Chinese
-
Abstract:
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 genomic 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 expressed product contained about 20% of total somatic soluble protein and reached a purity of 85% after purification . It was verified by the Western blot. Enzyme unit per millilitre of purified products was about 72. Conclusion 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.
