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Cloning of Essential Genes and Purification of Encoded Proteins in Escherichia coli

 

D. H. Gilling#  , and H. H. Xu

 

Department of Biological Sciences

California State University, Los Angeles

 

 

     Background:  With the emergence and spread of multi-drug resistant bacterial pathogens, there is an urgent yet unmet medical need for discovery and development of novel antibiotics.  Novel or under exploited bacterial essential target proteins represent valuable therapeutic targets for the discovery of novel antibacterial compounds.  The objective of this study is to clone bacterial essential genes into an expression vector and over-express and purify the encoded essential protein targets for biochemical high throughput screening and mechanism of action studies.

     Methods:  A ligation- independent cloning (LIC) vector (pET30Ek/LIC) was used to clone three essential genes from E. coli.  This vector allows high efficient cloning of genes and expression of histidine (His) tagged protein fusions for ease of purification process.  Gene sequences were PCR amplified by using Pfu DNA polymerase from Stratagene.  Cloned genes were confirmed via restriction enzyme digestion followed by agarose gel electrophoresis.  Over-expressed His tagged proteins were purified by using a nickel column kit from Novagen. Protein over-expression and purification were monitored by polyacrylamide gel electrophoresis. 

     Results:  Genes fabI (encoding enol-acyl carrier protein reductase), murA (encoding UDP-N-acetylglucosamine enolpyruvyl transferase), and trpS (encoding tryptophanyl tRNA synthetase) have all been cloned into vector pET30Ek/LIC in E. coli host strain BL21(DE3) as confirmed by restriction enzyme digest and agarose gel electrophoresis.  Cells of each clone were grown overnight with IPTG induction.  Cell free extracts were obtained and over-expression of three essential protein targets was confirmed by polyacrylamide gel electrophoresis.  Electrophoresis results indicate that for each cell clone one particular over-expressed protein band migrated to an area consistent with expected size of the protein encoded by the cloned essential gene.  Over-expressed His-tagged proteins were purified with the aid of the nickel column.  Impurities (imidazole) were removed via dialysis and centrifugation.  These purified essential proteins are being used in biochemical assays involving enzyme substrates and known inhibitors. 

     Conclusion:  Using a LIC vector, three essential genes from E. coli have been efficiently cloned and over-expressed.  The over-expressed proteins have been purified to near homogeneity.  These essential proteins are valuable reagents to screen for novel enzyme inhibitors as well as to study biochemical interactions with substrates and inhibitors.  

     Acknowledgements:  Funding for this project has been partially supported by NIH RIMI (P20MD001824) and NIH MBRS-SCORE grants (SGM008101) to H. H. Xu.  D. H. G. was supported through NIH MBRS RISE award R25 GM61331. We would like to thank both Lilian Real and David Yang for their technical assistance.