TY - JOUR T1 - Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding JO - Revista Argentina de Microbiología T2 - AU - Pavan,María Elisa AU - Pavan,Esteban Enrique AU - Cairó,Fabián Martín AU - Pettinari,María Julia SN - 03257541 M3 - 10.1016/j.ram.2015.10.004 DO - 10.1016/j.ram.2015.10.004 UR - https://www.elsevier.es/es-revista-revista-argentina-microbiologia-372-articulo-expression-refolding-protective-antigen-bacillus-S0325754115001509 AB - 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. ER -