Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase

We cloned and sequenced the murG gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences fro m different bacteria confirmed the presence of highly conserved regions hav ing sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The mur C gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL2I(lambda DE3), Three PCR cloning strategies a;ere used to obtai n the three recombinant plasmids for expression of the native MurC, MurC Hi s-ragged at N-terminal and at C-terminal, respectively, MurC His-ragged at C-terminal was chosen for large scale production and protein purification i n the soluble form. The purification was done in a single chromatographic s tep on an affinity nickel column and obtained in mg quantities at 95% homog eneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings, Detailed s tudies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for desig n of novel antibacterials. (C) 2000 Published by Elsevier Science B.V. All rights reserved.