Optimization and One-Step Purification of Recombinant V Antigen Production from Yersinia pestis
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ORIGINAL PAPER
Optimization and One‑Step Purification of Recombinant V Antigen Production from Yersinia pestis Elahe Seyed Hosseini1,2 · Mehdi Zeinoddini1,2 · Ali Reza Saeedinia2 · Valiollah Babaeipour2
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract The purpose of this study was to develop an efficient and inexpensive method for the useful production of recombinant protein V antigen, an important virulence factor for Yersinia pestis. To this end, the synthetic gene encoding the V antigen was subcloned into the downstream of the intein (INT) and chitin-binding domain (CBD) from the pTXB1 vector using specific primers. In the following, the produced new plasmid, pTX-V, was transformed into E. coli ER2566 strain, and the expression accuracy was confirmed using electrophoresis and Western blotting. In addition, the effects of medium, inducer, and temperature on the enhancement of protein production were studied using the Taguchi method. Finally, the V antigen was purified by a chitin affinity column using INT and CBD tag. The expression was induced by 0.05 mM IPTG at 25 °C under optimal conditions including TB medium. It was observed that the expression of the V-INT–CBD fusion protein was successfully increased to more than 40% of the total protein. The purity of V antigen was as high as 90%. This result indicates that V antigen can be produced at low cost and subjected to one-step purification using a self-cleaving INT tag. Keywords V antigen · Yersinia pestis · Intein · Expression · Purification · Optimization
Introduction Yersinia pestis has evolved from gastrointestinal pathogens and its antibiotic resistance can cause a dangerous disease, i.e., plague. Therefore, plague is still considered as a major threat to human health [1-3]. The human plague vaccine (USP) is a killed whole-cell plague bacilli that prevents plague infections through subcutaneous injection [4]. However, some reports have recently demonstrated the cytotoxic effects of whole-cell vaccines and their poor protection against virulent strains without capsules [5, 6]. The low calcium response (Lcr) of V antigen (LcrV) and the component 1 (F1) capsular antigen are the two important virulence factors which have been considered as vaccine candidates tested for their efficacy on humans and primates. LcrV is known as the virulence and multifunctional protein. This crucial protein has been shown to act at the * Mehdi Zeinoddini [email protected] 1
Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran
Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran
2
level of secretion control by binding to other proteins in order to modulate the host immune response by altering cytokine production [7, 8]. Genetic engineering can be used to produce recombinant vaccines using different parts of Yersinia, such as V antigen and F1 [9, 10]. To this purpose, the selection of effective methods to recombinant protein purification is a key factor of pro
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