Overproduction and purification of Escherichia coli tRNALeu

被引：39

作者：

Li, Y ^{[1
]}

Wang, ED ^{[1
]}

Wang, YL ^{[1
]}

机构：

[1] Chinese Acad Sci, Shanghai Inst Biochem, State Key Lab Mol Biol, Shanghai 200031, Peoples R China

来源：

SCIENCE IN CHINA SERIES C-LIFE SCIENCES | 1998年 / 41卷 / 03期

关键词：

tRNA(1)(Leu); tRNA(2)(Leu); cloning; high-expression; purification;

D O I：

10.1007/BF02895095

中图分类号：

Q [生物科学];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Chemically synthesized genes encoding Escherichia coli tRNA(1)(Leu) and rRNA(2)(Leu) were ligated into the plasmid pTrc99B, then transformed into Escherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucine accepting activity of their total tRNA reached 810 and 560 pmol/A(260), respectively: the content of tRNA(1)(Leu) was 50% of total tRNA from MT-Leu1, while that of tRNA(2)(Leu) was 30% of total tRNA from MT-Leu2. Both tRNA(Leu)s from their total tRNAs were fractionated to 1 600 pmol/A(260) after DEAE-Sepharose and ED-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of rRNA(Leu) catalyzed by leucyl-tRNA synthetase were determined.

引用

页码：225 / 231

页数：7