In vivo overexpression and purification of Escherichia coli tRNAser

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Abstract

DNA fragments corresponding to the sequences of Escherichia coli tRNA2ser and amber suppressor tRNAser, 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 tRNAser of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA2ser). From these systems 10 mg quantities of tRNAsers could be isolated with a serine acceptance of 1,100 pmol/A280 unit. © 1993.

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Borel, F., Härtlein, M., & Leberman, R. (1993). In vivo overexpression and purification of Escherichia coli tRNAser. FEBS Letters, 324(2), 162–166. https://doi.org/10.1016/0014-5793(93)81385-D

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