PCR MUTAGENESIS AND OVEREXPRESSION OF TRYPTOPHAN SYNTHASE FROM SALMONELLA-TYPHIMURIUM - ON THE ROLES OF BETA(2) SUBUNIT LYS-382

We have devised convenient methods for mutagenesis and very high level expression of wild type and mutant tryptophan synthase alpha and beta (2) subunits and alpha(2) beta(2) complex from Salmonella typhimurium, The trpBA genes were modified by introduction of five new restriction sites by polymerase chain reaction (PCR) and mere then cloned into th e plasmid pTrc99A under trc promoter control. The recombinant plasmid pEBA-10 and three plasmids constructed from pEBA-10 were transformed i nto Escherichia coli CB149, which lacks tryptophan operon genes. Optim ization of growth conditions of the transformed cells resulted in 10- to 40-fold higher yields of cells (similar to 22 g/liter) than attaine d previously. The improved expression system gave higher yields of try ptophan synthase proteins (23-70% of the soluble protein) and led to c orrespondingly high yields of purified alpha and beta(2) subunits or a lpha(2) beta(2) complex (200-800 mg/liter). A plasmid containing 8 cop ies of the trpA gene gave the highest yield of alpha subunit. The PCR- based mutagenesis method permits mutation of any base pair in the trpB A genes, between suitable pairs of restriction sites, and requires onl y one new primer per mutation, The method is illustrated by constructi on of mutant beta(2) subunits with any of five amino acid substitution s at Lys-382, the site of a previously described missense mutation. Ch aracterization of the purified mutant alpha(2) beta(2) complexes shows that Lys-382 in the wild type alpha(2) beta(2) complex does not serve an essential catalytic role but may stabilize an active ''closed'' co nformation of the enzyme by forming a salt bridge with Glu-350.