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

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 overexpressed antigenic proteins.

Atomic Structures of Anthrax Prechannel Bound with Full-Length Lethal and Edema Factors

“…Anthrax is an ancient and deadly disease caused by the sporeforming bacterial pathogen Bacillus anthracis (Doganay and Demiraslan, 2015;Pavan et al, 2011). Today anthrax mostly affects wildlife and livestock, although it remains a major public health concern, particularly among people who handle contaminated animal products (Pohanka, 2019).…”

Protective antigen domain 4 of <i>Bacillus anthracis</i> as a candidate for use as vaccine for anthrax

“…The expression plasmids were used to transform competent E. coli BL21Star (DE3)pLys One Shot to obtain expression clones verified by sequencing. The high expression of PA obtained by Pavan and et al combined with the low background and the enrichment of PA in the lysis step, facilitated PA purification using only washes, without the need for chromatographic purification steps [15].…”

CLPC1 protein inhibition, antimycobacterial, and anti‐inflammatory properties of the metabolite from Streptomyces wuyuanensis collected in Nghe An province, Vietnam

“…When OD value of the culture was reached to 0.6-0.8 at 600 nm, the concentration of bacteria in culture medium was determined and the protein was expressed by IPTG (isopropyl01-thio-β-D galactopyranosidee-Sigma). 23,24 Cell residue was recovered by centrifugation and was broken down the cell membrane by ultrasound method. The recombinant ClpC1 protein was purified using Ni-TED spin column (Macherey-Nagel).…”