Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding
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 c...
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2016
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03257541_v48_n1_p5_Pavan http://hdl.handle.net/20.500.12110/paper_03257541_v48_n1_p5_Pavan |
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paper:paper_03257541_v48_n1_p5_Pavan2023-06-08T15:32:56Z Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding 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 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. 2016 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03257541_v48_n1_p5_Pavan http://hdl.handle.net/20.500.12110/paper_03257541_v48_n1_p5_Pavan |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding |
| topic_facet |
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 |
| description |
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. |
| title |
Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding |
| title_short |
Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding |
| title_full |
Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding |
| title_fullStr |
Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding |
| title_full_unstemmed |
Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding |
| title_sort |
expression and refolding of the protective antigen of bacillus anthracis: a model for high-throughput screening of antigenic recombinant protein refolding |
| publishDate |
2016 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03257541_v48_n1_p5_Pavan http://hdl.handle.net/20.500.12110/paper_03257541_v48_n1_p5_Pavan |
| _version_ |
1768545144039014400 |