OVERPRODUCTION AND ONE-STEP PURIFICATION OF ESCHERICHIA-COLI UDP-N-ACETYLGLUCOSAMINE ENOLPYRUVYL REDUCTASE

The Escherichia coli gene murB, encoding the enzyme tyl-2-amino-2-deox y-3-O-lactylglucose:nicotinamide adenine dinucleotide phosphate oxidor eductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the pepti doglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. Th e synthetic gene was subcloned into the NdeI and BamHI restriction sit es of the expression vector pT7-7, designed to utilize T7 RNA polymera se to direct transcription of the target gene, in a two-step procedure , The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give t he plasmid pT7-7-murB-590. The construction of the desired overproduci ng plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricte d pT7-7-murB-590 plasmid followed by restriction digestion to select t he properly oriented insert, pT7-7-murB. Overexpression of EP-reductas e from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein pe r 3 wet grams of E. coli cells, The EP-reductase was purified in a sin gle step utilizing dye-ligand chromatography to yield 30 mg of pure pr otein, The availability of these levels of reductase will allow the me chanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition , the EP-reductase generated in this study has been utilized as a coup ling enzyme to assay the first enzyme in the peptidoglycan biosyntheti c pathway, UDP-N-acetyl-glucosamine enolpyruvyl transferase, and these results are also presented. (C) 1995 Academic Press, Inc.