DNA fragments corresponding to the sequences of Escherichia coli tRNA2 ser 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 tRNA2 ser). From these systems 10 mg quantities of tRNAsers could be isolated with a serine acceptance of 1,100 pmol/A 280 unit.
