INVIVO OVEREXPRESSION AND PURIFICATION OF ESCHERICHIA-COLI TRANSFER RNA(SER)

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 t ranscript of the correct size. The genes of promotor, fragment and ter minator 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(se r) 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 tRNA(ser)s could be isolated with a serin e acceptance of 1,100 pmol/A280) unit.