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In vivo overexpression and purification of Escherichia coli tRNA ser
Author(s) -
Borel Franck,
Härtlein Michael,
Leberman Reuben
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81385-d
Subject(s) - escherichia coli , transfer rna , in vivo , chemistry , biochemistry , microbiology and biotechnology , biology , genetics , gene , rna
DNA fragments corresponding to the sequences of Escherichia coli tRNA 2 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 tRNA 2 ser ). From these systems 10 mg quantities of tRNA ser s could be isolated with a serine acceptance of 1,100 pmol/ A 280 unit.