Overexpression and purification of the separate tryptophan synthase α and β subunits from Salmonella typhimurium

Abstract

To obtain high levels of expression of the free α and β subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the α and β subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual α and β subunits. Microcrystals of the β subunit are obtained by addition of polyethylene glycol 5000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase α 2 β 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the α subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified α and β subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate α and β subunits. The purified α and β subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.