Abstract
Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein's functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.
Highlights
Bacillus anthracis, the causative agent of the disease anthrax, has two major virulence factors, one in the form of exotoxins and the other in the form of unique capsule, which are encoded by the plasmids pXO1 and pXO2, respectively [1, 2]
Due to the risk associated with the handling of B. anthracis culture for production and purification of native protective antigen (PA) from bacterial culture supernatant, various laboratories have opted for the production of recombinant PA expressed conveniently in E. coli host
We have optimized the growth parameters for E. coli to obtain a maximum production of recombinant PA in soluble form from three different expression vectors
Summary
The causative agent of the disease anthrax, has two major virulence factors, one in the form of exotoxins and the other in the form of unique capsule, which are encoded by the plasmids pXO1 and pXO2, respectively [1, 2]. AVA and AVP are effective, their undefined composition, batch to batch variation, extensive dosing regimen, and adverse immunological side effects have made way for the search of secondgeneration anthrax vaccine containing recombinant PA that is under developmental stage [21, 22] These recombinant vaccines differ from their predecessors in that their composition will be defined, amenable to large scale production, and will be free from any adverse side effects. Apart from E. coli, numerous other attempts to develop expression systems are based on variety of organisms including attenuated strains of B. anthracis, B. subtilis, B. brevis, and Salmonella typhimurium [21, 24,25,26] These approaches are not simpler and often require multiple harsh purification steps which may result in reduced yield and stability [27, 28]. This study highlights the straightforward production of large quantity of recombinant PA in its biologically active soluble form using an optimized E. coli vector-host combination system
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