Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding

PAVAN, M. ELISA; PAVAN ESTEBAN E; CAIRO, FABIAN; M. JULIA PETTINARI

REVISTA ARGENTINA DE MICROBIOLOGíA

ASOCIACION ARGENTINA MICROBIOLOGIA

Lugar: Buenos Aires; Año: 2016 vol. 48 p. 5 - 14

0325-7541

Abstract 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.