Gene Cloning, Overproduction and Purification of Escherichia coli tRNA(Arg)(2).
A synthetic gene encoding Escherichia coli tRNA(Arg)(2) was inserted in a plasmid under the control of an isopropyl-beta, D-thiogalactopyranoside (IPTG)-inducible promotor, pTrc99B. In E.coli MT102 transformed by the above plasmid containing the target gene. TRNA(Arg)(2) was overproduced up to 30 fold of that of the host. In the transformant the quantity contained tRNA(Arg) increased 10 times and was 70% of the total tRNA. The tRNA(Arg)(2) was purified to 88% homogeneity by passing through a DEAE-Sephacel column, and then was purified by benzyl-DEAE cellulose column chromatography to a purity of 99% with an arginylation activity of 1 600 pmole/A(260) unit. Eighteen milligrams of tRNA(Arg)(2) could be obtained from 40 mg total tRNA which was obtained from four liters of overnight culture, and the yield of the purification was 62%. The accurate kinetic constants of aminoacylation of tRNA(Arg)(2) catalyzed by arginyl-tRNA synthetase were comparable with that of tRNA(Arg) from Sigma.
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. wed@server.shcnc.ac.cn
Jin-Fu
JF
Wu
En-Duo
ED
Wang
Ying-Lai
YL
Wang
Eriani
E
Gilbert
Gangloff
G
Jean