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Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate
A El Zoeiby1, F Sanschagrin, J Lamoureux
1Centre de recherche sur la fonction, structure et ingénierie des protéines, Faculté de médecine, pavillon Charles-Eugène-Marchand, Ste-Foy, Que., Canada.
Abstract:
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.
Insights
Researchers cloned and sequenced the murC gene from Pseudomonas aeruginosa, successfully expressing and purifying the MurC protein. This work provides essential tools for developing novel antibacterial agents.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The murC gene is essential for bacterial cell wall synthesis.
- Pseudomonas aeruginosa is an opportunistic pathogen requiring novel therapeutic strategies.
- Understanding murC function is crucial for developing new antibiotics.
Purpose of the Study:
- To clone, sequence, and express the murC gene from Pseudomonas aeruginosa.
- To produce purified recombinant MurC protein for antibody generation and assay development.
- To facilitate high-throughput screening for novel antibacterial drug discovery.
Main Methods:
- Gene cloning and sequencing of murC from Pseudomonas aeruginosa.
- Expression of recombinant MurC protein in Escherichia coli using the pET30a vector.
- Purification of His-tagged MurC protein using affinity chromatography.
- Production of monoclonal antibodies against purified MurC protein.
Main Results:
- The murC gene was successfully cloned and sequenced.
- Recombinant MurC protein, particularly the C-terminal His-tagged version, was overexpressed and purified to 95% homogeneity.
- Functional complementation in E. coli (murCts) demonstrated the gene's essentiality.
- Monoclonal antibodies were generated for MurC.
Conclusions:
- The successful cloning, expression, and purification of Pseudomonas aeruginosa murC provide valuable reagents for further research.
- This study lays the groundwork for developing high-throughput screening assays to identify new antibacterial compounds targeting MurC.
- The generated antibodies and purified protein are crucial for epitope mapping and understanding bacterial cell cycle regulation.