Cloning, expression, purification and activity assay of Trypanosoma brucei phenylalanyl-tRNA synthetase in Escherichia coli.
- Author:
Ying YAO
1
;
Guangwei GAO
;
Dawei LI
Author Information
1. School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Phenylalanine-tRNA Ligase;
biosynthesis;
genetics;
Protozoan Proteins;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Trypanosoma brucei brucei;
enzymology;
genetics
- From:
Chinese Journal of Biotechnology
2010;26(1):130-135
- CountryChina
- Language:Chinese
-
Abstract:
Phenylalany--tRNA synthetase is a key enzyme for protein synthesis in Trypanosoma. Its validation as an inhibition. target will enable the development of a new generation of anti-Trypanosoma drugs. However, little is known about the isolation of the Trypanosoma Phenylalanyl-tRNA synthetase. Here we report the cloning, expression, purification, and activity assay of Phenylalanyl-tRNA synthetase from Trypanosoma brucei in Escherichia coli host. We co-cloned the alpha-subunit and beta-subunit of Phenylalanyl-tRNA synthetase from Trypanosoma brucei genomic DNA into the co-expression vector pCOLADuet. We successfully expressed the Trypanosoma brucei Phenylalanyl-tRNA synthetase in E. coli host, purified the whole enzyme by Ni-Hind affinity column and verified it by Western blotting. In addition, we tested its enzymatic activity by isotope labeling. The whole work laid a solid foundation for in vitro the screening and optimization of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitors.
