Cloning, expression, purification and activity assay of Trypanosoma brucei phenylalanyl-tRNA synthetase in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 130-135, 2010.
Artigo
em Chinês
| WPRIM
| ID: wpr-336251
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Fenilalanina-tRNA Ligase
/
Trypanosoma brucei brucei
/
Proteínas Recombinantes
/
Proteínas de Protozoários
/
Clonagem Molecular
/
Escherichia coli
/
Vetores Genéticos
/
Genética
/
Metabolismo
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2010
Tipo de documento:
Artigo
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