Optimization and One-Step Purification of Recombinant V Antigen Production from Yersinia pestis.

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 ER 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.