Degree Granting Department
My Lien Dao.
Metalloenzyme, Collagen, Pathogenic factor, Recombinant protein, Nosocomial infections
Bacillus anthracis and B. cereus are well known etiological agents, which cause disease in healthy and immunocompromised individuals. Considering the abundance and lethality of these organisms it is imperative that research is performed to identify and analyze new factors that may contribute to their pathogenicity. Camelysin is a membrane bound, zinc metalloprotease isolated from B. cereus. Assays performed on purified camelysin demonstrate that the protease exhibits fibrinolytic, collagenolytic, and actin degradation activity, any of which can contribute to the organisms ability to invade host tissues and cause damage. Considering the putative role of camelysin in pathogenicity, it would be beneficial to study the effects of camelysin in tissue cultures or animal models. The goal of this study focused on the cloning and expression of camelysin from B. cereus and its homolog in B. anthracis.
Expression of a fusion tagged protein may assist in the purification of camelysin as well as overcoming the native proteins extreme insolubility. Primers were designed to amplify the camelysin gene from B. cereus for cloning into the prokaryotic pBAD TOPO[registered trademark] TA, pET100/D-TOPO[registered trademark], and the eukaryotic pcDNA3.1/V5-His[copyright] TOPO[registered trademark] TA expression vectors. Primers were also designed to amplify the gene from B. anthracis for cloning into the pBAD TOPO[registered trademark] TA vector. The recombinant clones were induced and successful expression of the protein was confirmed by performing SDS-PAGE, Western blotting, and an azocasein protease assay. The recombinant proteins exhibited casein degradation activity which is observed with purified camelysin from B. cereus. This study successfully demonstrated the presence of the camelysin protein in B. anthracis.
Scholar Commons Citation
Myers, Andrew Ross, "Cloning, expression, and sequence analysis of camelysin, a zinc metalloprotease from bacillus anthracis and b. cereus" (2005). Graduate Theses and Dissertations.