Cloning of Essential Genes and Purification of Encoded Proteins in Escherichia coli
Damian H. Gilling and H. Howard Xu
Department of Biological Sciences, California State University at 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. In this study, several Escherichia coli essential genes were cloned into an expression vector, followed by the over-expression and purification of the encoded essential proteins for biochemical assays and drug binding studies using Surface Plasmon Resonance (SPR).
Methods: A ligation-independent cloning (LIC) vector (pET30Ek/LIC) was used to clone three essential genes from E. coli. This vector allows highly efficient cloning of genes and expression of histidine (His) tagged protein fusions which facilitate the purification of the over-expressed proteins. 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. The target-compound interactions are monitored via Surface Plasmon Resonance (SPR). Purified E. coli proteins were immobilized to a SPR chip surface and an inhibitor was then washed over. The relative binding between an inhibitor and a protein was recorded by SPR as response units (RUs).
Results: Essential 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 cells as confirmed by restriction enzyme digest and agarose gel electrophoresis. Over-expression of three essential proteins (drug targets) was confirmed by polyacrylamide gel electrophoresis. Over-expressed His-tagged proteins were purified with the aid of the nickel column. Impurities (imidazole) were removed via dialysis and centrifugation. The purified proteins have been successfully immobilized to SPR chips and target-inhibitor binding was observed using SPR.
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. Gilling was supported through NIH MBRS RISE award R25 GM61331. We would like to thank both Lilian Real and Ming Du for their technical assistance.