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
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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
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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
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Volumen: | 48
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Número: | 1
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Página de inicio: | 5
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Página de fin: | 14
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DOI: |
http://dx.doi.org/10.1016/j.ram.2015.10.004 |
Título revista: | Revista Argentina de Microbiologia
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Título revista abreviado: | Rev. Argent. Microbiol.
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ISSN: | 03257541
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CODEN: | RAMID
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CAS: | Antigens, Bacterial
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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