title: Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.
abstract: Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.
authors.affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; Biochemiq S.A., Laboratorio de Biología Molecular, Buenos Aires, Argentina.
authors.forename: María Elisa
authors.initials: ME
authors.lastname: Pavan
authors.affiliation: Laboratorio di Tecnologie Biomediche, Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Italy.
authors.forename: Esteban Enrique
authors.initials: EE
authors.lastname: Pavan
authors.affiliation: Biochemiq S.A., Laboratorio de Biología Molecular, Buenos Aires, Argentina; Facultad de Ciencias Veterinarias, UBA, Argentina.
authors.forename: Fabián Martín
authors.initials: FM
authors.lastname: Cairó
authors.affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; IQUIBICEN, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. Electronic address: jul@qb.fcen.uba.ar.
authors.forename: María Julia
authors.initials: MJ
authors.lastname: Pettinari
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000276:immunology*; D001408:Bacillus anthracis / Q000276:immunology* / Q000378:metabolism*; D008958:Models, Molecular; D058849:Protein Refolding
chemical_list: D000942:Antigens, Bacterial
keywords: Antígeno protector; Bacillus anthracis; Evaluación de alto rendimiento del replegado; High-throughput refolding screening; Protective antigen; Protein refolding; Replegado de proteínas