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Over-expression and Purification of full-length TcdE and Met27 TcdE Isoform for Structural Studies by NMR Spectroscopy


The bacterium Clostridium difficile or C. diff is the cause of Clostridium difficile-associated infection (CDI). CDI is a growing health concern in hospitals and health clinics. Proteins of pathogenic strains of C. diff have the potential of being pharmaceutical drug targets as there are currently no effective treatment or drug combination that eliminate infection with high efficacy. Incidences of

relapse for CDI are as high as 20% for first recurrence and increases with subsequent relapses [17, 36, 49, 71].

Though some companies and hospitals have experimented with fecal transplant as a treatment, this procedure is still classified as an Investigational New Drug and is considered by most insurance companies to be an uncovered experimental treatment [17]. While almost all transcription factors that lie within the pathogenicity locus as well as the proteins they encode have been well-characterized, the TcdE protein remains less understood though sequence homology studies suggest it may function is a holin-like protein to allow the selective release of toxins TcdA and TcdB during pathogenesis [8, 37, 39, 40, 45, 69]. Elucidating the structure of the protein may provide clues as to how C. diff pathogenicity progresses and may offer a new target for therapeutic drugs. The structure of membrane proteins of similar size (21kDa) and transmembrane helices have been successfully studied and solved using nuclear magnetic spectroscopy (NMR). As with structural studies of membrane proteins by NMR, the lipid environment chosen is important in guiding protein folding and its local dynamics. Past studies have shown that micellar environments can distort membrane protein structures while bicelles and bilayers provide environments that more closely recapitulate native environments.

All proteins behave differently in different lipid environments and not all proteins are suitable for studies by NMR as it requires the protein to be over-expressed, pure, monomeric and to fold and behave in lipids. Here, a proof-of-

concept is demonstrated that TcdE can be successfully over-expressed and reconstituted in bicelles for studies by solution- and solid-state NMR.

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