Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.
Rev Argent Microbiol
; 48(1): 5-14, 2016.
Article
em En
| MEDLINE
| ID: mdl-26777581
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bacillus anthracis
/
Modelos Moleculares
/
Redobramento de Proteína
/
Antígenos de Bactérias
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Revista:
Rev Argent Microbiol
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Argentina
País de publicação:
Argentina