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- W2079488552 abstract "We have devised convenient methods for mutagenesis and very high level expression of wild type and mutant tryptophan synthase α and β2subunits and α2β2complex fromSalmonella typhimurium.ThetrpBA genes were modified by introduction of five new restriction sites by polymerase chain reaction (PCR) and were then cloned into the plasmid pTrc99A under trc promoter control. The recombinant plasmid pEBA-10 and three plasmids constructed from pEBA-10 were transformed intoEscherichia coliCB149, which lacks tryptophan operon genes. Optimization of growth conditions of the transformed cells resulted in 10- to 40-fold higher yields of cells (∼22 g/liter) than attained previously. The improved expression system gave higher yields of tryptophan synthase proteins (23–70% of the soluble protein) and led to correspondingly high yields of purified α and β2subunits or α2β2complex (200–800 mg/liter). A plasmid containing 8 copies of thetrpA gene gave the highest yield of α subunit. The PCR-based mutagenesis method permits mutation of any base pair in thetrpBA genes, between suitable pairs of restriction sites, and requires only one new primer per mutation. The method is illustrated by construction of mutant β2subunits with any of five amino acid substitutions at Lys-382, the site of a previously described missense mutation. Characterization of the purified mutant α2β2complexes shows that Lys-382 in the wild type α2β2complex does not serve an essential catalytic role but may stabilize an active “closed” conformation of the enzyme by forming a salt bridge with Glu-350." @default.
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- W2079488552 title "PCR Mutagenesis and Overexpression of Tryptophan Synthase fromSalmonella typhimurium:On the Roles of β2Subunit Lys-382" @default.
- W2079488552 doi "https://doi.org/10.1006/prep.1996.0082" @default.
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