|Line, John - Eric|
Submitted to: ASM Conference
Publication Type: Abstract Only
Publication Acceptance Date: 2/21/2013
Publication Date: N/A
Technical Abstract: Expression and Characterization of Recombinant Campylobacter jejuni Chemotactic Proteins Hung-Yueh Yeh*, Kelli L. Hiett, John E. Line, Brian B. Oakley and Bruce S. Seal, Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, 950 College Station Road, Athens, GA 30605-2720. Background: Campylobacter jejuni is one of the leading etiologic agents of human acute bacterial gastroenteritis worldwide. Risk assessment studies have demonstrated that the source for human infection has been linked to consumption and handling of poultry. Reduction of this microorganism on poultry carcasses or during production results in reduction of human cases. Although C. jejuni has been extensively studied, there are many unanswered questions regarding pathogenesis and host immune responses. Because chemotaxis can guide bacteria to colonization sites, proteins involved in chemotaxis could be targets for vaccine development. In this study, we describe construction, expression and characterization of C. jejuni chemotactic proteins in a bacterial expression system. Methods: Genomic DNA from C. jejuni D1-39 was isolated, and chemotactic genes were amplified by PCR. The Expresso™ T7 Cloning and Expression kit was used to construct and express the recombinant (His-tagged) chemotactic proteins. Individually expressed proteins were separated by SDS-PAGE and detected by a Nickel HisDetector Western blot. These proteins were further characterized by MALDI-TOF analysis. Each recombinant protein was over-expressed in the presence of 1.0 mM IPTG in 0.5-liter LB broth and purified by a cobalt-chelating resin. The recombinant proteins were tested for their antigenicity by using sera from chickens experimentally inoculated with C. jejuni. Results: Twenty-two chemotactic genes were identified in the annotated C. jejuni genome sequence deposited in GenBank (accession no. CP000538). Fifteen genes were PCR amplified, sequenced and cloned from C. jejuni D1-39 genomic DNA. They were successfully expressed in the E. coli BL21(DE3) host cells. All recombinant proteins had the expected sizes on Coomassie-stained SDS-PAGE, and contained a six-His tag. We also observed the optical density of seven out of 22 bacterial cultures wherein proteins were not detected on the SDS-PAGE or in decreased amounts that leveled off after addition of IPTG, suggesting that the proteins are toxic to the E. coli host cells.