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Chinese Journal of Biotechnology ; (12): 130-135, 2010.
Article in Chinese | 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.


Subject(s)
Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Phenylalanine-tRNA Ligase , Genetics , Protozoan Proteins , Genetics , Recombinant Proteins , Genetics , Metabolism , Trypanosoma brucei brucei , Genetics
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