Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems

Yersinia pestis, the causative organism of plague, produces V antigen (LcrV ), a bifunctional protein with regulatory and virulence roles that has been shown to be highly protective against a plague challenge. A combined sub-u nit vaccine, comprising recombinant V and Fraction 1 antigens is currently being developed. We report here the expression and purification of recombin ant V antigen (rV) using three different expression systems: the N-terminal GST fusion pGEX-5X-2 and pGEX-6P-2 systems from Pharmacia Biotech, and the C-terminal CBD fusion (IMPACT I(TM)) system from New England Biolabs. Afte r cleavage from the carrier protein, the yields of rV were 25 mg l(-1) (pGE X-5X-2), 31 mg l(-1) (pGEX-6P-2) and 0.75 mg l(-1) (IMPACT I(TM)). All of t he recombinant proteins were immunogenic in mice, although there were some differences in their protective efficacy against subcutaneous challenge wit h Y. pestis. Whilst rV antigen derived from the IMPACT I(TM) and pGEX-6P-2 systems and given in two immunising doses protected fully against challenge with 1 x 10(7) colony forming units (cfu) of Y. pestis, there was breakthr ough in protection against 1 x 10(5) cfu of Y. pestis in animals immunised twice with rV from the pGEX-5X-2 system. From this study, the pGEX-6P-2 has been selected for the production of rV as a vaccine component. The pGEX-6P -2 system utilises a GST tagged PreScission(TM) Protease (a recombinant hum an rhinovirus 3C protease) to cleave the fusion protein, thereby allowing e fficient removal of the enzyme from the final product. In addition, the enz yme is not of animal origin, therefore making it suitable for vaccine produ ction. (C) 1999 Elsevier Science Ltd. All rights reserved.