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 immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli...
| Autores: | , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | Argentina |
| Institución: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Idioma: | inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/48529 |
| Acceso en línea: | http://hdl.handle.net/11336/48529 |
| Access Level: | acceso abierto |
| Palabra clave: | Bacillus anthracis Protective antigen Protein refolding High-throughput refolding screening https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| Sumario: | Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, whichfacilitated its efficient purification by simple washing steps; however, it could not be recognizedby specific antibodies. Refolding conditions were subsequently analyzed in a high-throughputmanner that enabled nearly a hundred different conditions to be tested simultaneously. Therecovery of the ability of PA to be recognized by antibodies was screened by dot blot usinga coefficient that provided a measure of properly refolded protein levels with a high degreeof discrimination. The best refolding conditions resulted in a tenfold increase in the intensityof the dot blot compared to the control. The only refolding additive that consistently yieldedgood results was L-arginine. The statistical analysis identified both cooperative and negativeinteractions between the different refolding additives. The high-throughput approach describedin this study that enabled overproduction, purification and refolding of PA in a simple andstraightforward manner, can be potentially useful for the rapid screening of adequate refoldingconditions for other overexpressed antigenic proteins. |
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