title: Purification and characterization of Yersinia enterocolitica and Yersinia pestis LcrV-cholera toxin A(2)/B chimeras.
abstract: Yersinia pestis is a virulent human pathogen and potential biological weapon. Despite a long history of research on this organism, there is no licensed vaccine to protect against pneumonic forms of Y. pestis disease. In the present study, plasmids were constructed to express cholera toxin A(2)/B chimeric molecules containing the LcrV protective antigen from Yersinia enterocolitica and Y. pestis. These chimeras were expressed and purified to high yields from the supernatant of transformed Escherichia coli. Western and GM(1) ELISA assays were used to characterize the composition, receptor-binding and relative stability of the LcrV-CTA(2)/B chimera in comparison to cholera toxin. In addition, we investigated the ability of the Y. pestis LcrV-CTA(2)/B chimera to bind to and internalize into cultured epithelial cells and macrophages by confocal microscopy. These studies indicate that the uptake and trafficking of the LcrV antigen from the chimera is comparable to the trafficking of native toxin. Together these findings report that stable, receptor-binding, non-toxic LcrV-cholera toxin A(2)/B chimeras can be expressed at high levels in E. coli and purified from the supernatant. In addition, the internalization of antigen in vitro reported here supports the development of these molecules as novel mucosal vaccine candidates.
authors.affiliation: Department of Biological Sciences, Boise State University, Boise, ID 83725, USA. juliettetinker@boisestate.edu
authors.forename: Juliette K
authors.initials: JK
authors.lastname: Tinker
authors.forename: Chadwick T
authors.initials: CT
authors.lastname: Davis
authors.forename: Britni M
authors.initials: BM
authors.lastname: Arlian
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D001428:Bacterial Vaccines / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D002772:Cholera Toxin / Q000235:genetics* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D010930:Plague / Q000517:prevention & control; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics* / Q000302:isolation & purification*; D015009:Yersinia Infections / Q000235:genetics; D015008:Yersinia enterocolitica / Q000235:genetics; D015010:Yersinia pestis / Q000235:genetics*
chemical_list: D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; D002772:Cholera Toxin