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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 over expressed antigenic proteins. © 2015 Asociación Argentina de Microbiología.

Registro:

Documento: Artículo
Título:Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
Autor:Pavan, M.E.; Pavan, E.E.; Cairó, F.M.; Pettinari, M.J.
Filiación:Departamento de Química Biológica, Universidad de Buenos Aires, Argentina
Biochemiq S.A, Laboratorio de Biología Molecular, Buenos Aires, Argentina
Laboratorio di Tecnologie Biomediche, Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Italy
UBA, Argentina
IQUIBICEN, Universidad de Buenos Aires, Argentina
Palabras clave:Bacillus anthracis; High-throughput refolding screening; Protective antigen; Protein refolding; bacterial antigen; bacterial antigen; antigen expression; Article; Bacillus anthracis; dot hybridization; high throughput screening; molecular cloning; nonhuman; polymerase chain reaction; protein expression; protein purification; protein refolding; Bacillus anthracis; biosynthesis; chemical structure; immunology; metabolism; Antigens, Bacterial; Bacillus anthracis; Models, Molecular; Protein Refolding
Año:2016
Volumen:48
Número:1
Página de inicio:5
Página de fin:14
DOI: http://dx.doi.org/10.1016/j.ram.2015.10.004
Título revista:Revista Argentina de Microbiologia
Título revista abreviado:Rev. Argent. Microbiol.
ISSN:03257541
CODEN:RAMID
CAS:Antigens, Bacterial
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03257541_v48_n1_p5_Pavan

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Citas:

---------- APA ----------
Pavan, M.E., Pavan, E.E., Cairó, F.M. & Pettinari, M.J. (2016) . Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding . Revista Argentina de Microbiologia, 48(1), 5-14.
http://dx.doi.org/10.1016/j.ram.2015.10.004
---------- CHICAGO ----------
Pavan, M.E., Pavan, E.E., Cairó, F.M., Pettinari, M.J. "Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding " . Revista Argentina de Microbiologia 48, no. 1 (2016) : 5-14.
http://dx.doi.org/10.1016/j.ram.2015.10.004
---------- MLA ----------
Pavan, M.E., Pavan, E.E., Cairó, F.M., Pettinari, M.J. "Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding " . Revista Argentina de Microbiologia, vol. 48, no. 1, 2016, pp. 5-14.
http://dx.doi.org/10.1016/j.ram.2015.10.004
---------- VANCOUVER ----------
Pavan, M.E., Pavan, E.E., Cairó, F.M., Pettinari, M.J. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding . Rev. Argent. Microbiol. 2016;48(1):5-14.
http://dx.doi.org/10.1016/j.ram.2015.10.004