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 wild-type, 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.
Crouvoisier
M
M
Biochimie Structurale et Cellulaire, CNRS, Université Paris-Sud, Orsay, France.
Mengin-Lecreulx
D
D
van Heijenoort
J
J
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D005816:Genetic Complementation Test; D006639:Histidine; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase