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-termin al His-tagged MurG N-acetylglucosaminyl transferase from Escherichia coli w ere constructed. In complementation tests the three forms were active in vi vo. After IPTG induction, growth, spheroplast formation and lysis, overprod uced MurG proteins were mainly present (90%) in the particulate fraction. R eadily solubilized by CHAPS, they were purified without any detergent to ov er 80% purity for both His-tagged forms but only up to 20% for the wild-typ e form. The enzymatic activity of each purified MurG protein was determined and found to be inhibited to the same extent by ramoplanin, (C) 1999 Feder ation of European Biochemical Societies.