Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.
We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having 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 murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.
El Zoeiby
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A
Centre de recherche sur la fonction, structure et ingénierie des protéines, Faculté de médecine, pavillon Charles-Eugène-Marchand, Ste-Foy, Que., Canada.
Sanschagrin
F
F
Lamoureux
J
J
Darveau
A
A
Levesque
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase