Gene Cloning

Gene Cloning, Overproduction and
Purification of Escherichia coli tRNA^Arg₂

WU Jin-Fu, WANG En-Duo^* and WANG Ying-Lai
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China
)
Gilbert Eriani and Jean Gangloff
( Institut de biologie Moleculaire et Cellulaire du CNRS, 67084 Strasbourg,
France )

Abstract　　A synthetic gene encoding
Escherichia coli tRNA^Arg₂ was inserted in a plasmid under the
control of an isopropyl-β, D-thiogalactopyranoside (IPTG)-inducible promotor,
pTrc99B. In E.coli MT102 transformed by the above plasmid containing
the target gene. tRNA^Arg₂ 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^Arg₂  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/A₂₆₀ unit. Eighteen
milligrams of tRNA^Arg₂  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^Arg₂ catalyzed by arginyl-tRNA
synthetase were comparable with that of tRNA Arg from Sigma.
Key Words　　tRNA^Arg₂ ； cloning； overproduction； purification

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