Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.
To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta 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 alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 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 alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta 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 alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.
Yang
X J
XJ
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Ruvinov
S B
SB
Miles
E W
EW
D000595:Amino Acid Sequence; D001483:Base Sequence; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*; D014367:Tryptophan Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014367:Tryptophan Synthase