UDP-N-acetylglucosamine:: N-acetylmuramoyl-(pentapeptide) pyrophosphoryl undecaprenol N-acetylglucosamine transferase from Escherichia coli:: overproduction, solubilization, and purification

Plasmids for the high-level overproduction of wildtype, and C- and N-terminal His-tagged MurG N-acetylglucosaminyl transferase from Escherichia coli were constructed. In complementation tests the three forms were active in vivo. After IPTG induction, growth, spheroplast formation and lysis, overproduced MurG proteins were mainly present (90%) in the particulate fraction. Readily solubilized by CHAPS, they were purified without any detergent to over 80% purity for both His-tagged forms but only up to 20% for the wild-type form. The enzymatic activity of each purified MurG protein was determined and found to be inhibited to the same extent by ramoplanin, (C) 1999 Federation of European Biochemical Societies.