title: In vivo overexpression and purification of Escherichia coli tRNA(ser).
abstract: DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) 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 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees 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(2ser)). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit.
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, France.
authors.forename: F
authors.initials: F
authors.lastname: Borel
authors.forename: M
authors.initials: M
authors.lastname: Härtlein
authors.forename: R
authors.initials: R
authors.lastname: Leberman
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012313:RNA; D012362:RNA, Transfer, Ser / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D012362:RNA, Transfer, Ser; C039919:RNA, recombinant; D012313:RNA