Very high level overexpression of bacterial elongation factor Tu

The tufA gene, which encodes the bacterial elongation factor Tu of E. coli, was isolated as a 1226 bp DNA fragment using the polymerase chain reaction(1). EcoR1 restriction enzyme digestion sites were incorporated at the 3' and 5' termini of the gene. The tufA gene was ligated into the plasmid vector pUC19 and was sequenced fully. A plasmid shown to contain the tufA gene in the correct orientation with respect to the lac promoter of pUC19 was then used to transform E.coli strain JM105 and expression of EFTu in induced cultures of the resulting clone, JM105 pJBDB01, was measured using a continuous monitoring procedure. The clone was shown to produce up to 95 mg of bacterial elongation factor Tu per litre of cell culture.