PCR mutagenesis and overexpression of tryptophan synthase from Salmonella typhimurium: on the roles of beta2 subunit Lys-382.
We have devised convenient methods for mutagenesis and very high level expression of wild type and mutant tryptophan synthase alpha and beta2 subunits and alpha2beta2 complex from Salmonella typhimurium. The trpBA 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 into Escherichia coli CB149, which lacks tryptophan operon genes. Optimization of growth conditions of the transformed cells resulted in 10- to 40-fold higher yields of cells ( approximately 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 alpha and beta2 subunits or alpha2beta2 complex (200-800 mg/liter). A plasmid containing 8 copies of the trpA gene gave the highest yield of alpha subunit. The PCR-based mutagenesis method permits mutation of any base pair in the trpBA genes, between suitable pairs of restriction sites, and requires only one new primer per mutation. The method is illustrated by construction of mutant beta2 subunits with any of five amino acid substitutions at Lys-382, the site of a previously described missense mutation. Characterization of the purified mutant alpha2beta2 complexes shows that Lys-382 in the wild type alpha2beta2 complex 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.
Yang
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Lh
Enzyme Structure and Function Section, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Ahmed
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SA
Miles
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EW
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D014367:Tryptophan Synthase