In vivo overexpression and purification of Escherichia coli tRNAser

FEBS Letters

Borel, Franck¹  Härtlein, Michael¹  Leberman, Reuben¹ 

[1] European Molecular Biology Laboratory, Grenoble Outstation, 156 X, 38042 Grenoble Cedex, France

关键词: Serine;    tRNA;    Aminoacyl-tRNA synthetase;    Overexpression;    BAPPS;    N;    N'-bis(3-aminopropyl)-piperazine substituted Sepharose;    BSA;    bovine serum albumin;    TBE;    tris borate EDTA buffer;    TCA;    trichloroacetic acid;   

DOI  :  10.1016/0014-5793(93)81385-D

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

DNA fragments corresponding to the sequences of Escherichia coli tRNA₂ ^ser and amber suppressor tRNA^ser, were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37°C this vector has a low copy number but, following a temperature shift to 42°C, the copy number is no longer regulated. Using these constructs an overexpression of tRNA^ser of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA₂ ^ser). From these systems 10 mg quantities of tRNA^sers could be isolated with a serine acceptance of 1,100 pmol/A ₂₈₀ unit.

【 授权许可】

Unknown