Overproduction and purification of Escherichia coli tRNALeu |
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Authors: | LI Yong WANG Enduo WANG Yinglai |
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Institution: | (1) Statc Key Labratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai, China |
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Abstract: | Chemically synthesized genes encodingEscherichia coli tRNA
1
Leu
and tRNA
2
Leu
were ligated into the plasmid pTrc99B. then transformed intoEscherichia 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, leucinc accepting activity of their total tRNA reached
810 and 560 pmol/A260, 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 tRNALeus from their rotal tRNs were fractionated to 1 600 pmol/A260 after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two
isoacceptors of tRNALeu catalyzed by leucyl-tRNA synthetase were determined.
Project supported by the National Natural Science Foundation of China (Grant No. 39570164). |
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Keywords: | tRNA 1 Leu tRNA 2 Leu clonlng high-expression purification |
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