布氏锥虫苯丙氨酰-tRNA合成酶在大肠杆菌中的克隆、表达、纯化及活性测定

Cloning, expression, purification and activity assay of Trypanosoma brucei phenylalanyl-tRNA synthetase in Escherichia coli

摘要:

苯丙氨酰-tRNA合成酶是布氏锥虫蛋白合成过程中的一类重要酶，以其为靶点的抑制剂可能发展成为新一代的抗锥虫药物，但此前并没有分离锥虫苯丙氨酸-tRNA合成酶的报道。本研究用大肠杆菌成功克隆表达并纯化了布氏锥虫苯丙氨酰-tRNA合成酶并进行了活性测定。首先通过PCR方法从布氏锥虫细胞基因组中分别扩增出苯丙氨酰-tRNA合成酶的α亚基、β亚基的基因，依次克隆入pCOLADuet共表达载体，然后在大肠杆菌BL21 (DE3) RIPL中进行了成功表达，并采用Ni-Bind亲和层析对其进行了纯化，最后用免疫印迹进行了鉴定。此外还采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苯丙氨酰-tRNA合成酶抑制物的设计和体外筛选奠定了良好的基础。

Abstract:

Phenylalanyl-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 α-subunit and β-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.