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-unit vaccine, comprising recombinant V and Fraction 1 antigens is currently being developed. We report here the expression and purification of recombinant 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) system from New England Biolabs. After cleavage from the carrier protein, the yields of rV were 25 mg l(-1) (pGEX-5X-2), 31 mg l(-1) (pGEX-6P-2) and 0.75 mg l(-1) (IMPACT I). All of the recombinant proteins were immunogenic in mice, although there were some differences in their protective efficacy against subcutaneous challenge with Y. pestis. Whilst rV antigen derived from the IMPACT I 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 breakthrough 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 Protease (a recombinant human rhinovirus 3C protease) to cleave the fusion protein, thereby allowing efficient removal of the enzyme from the final product. In addition, the enzyme is not of animal origin, therefore making it suitable for vaccine production.
Carr
S
S
Defence Evaluation and Research Agency, CBD, Salisbury, Wiltshire, UK. 100432.3200@compuserve.com
Miller
J
J
Leary
S E
SE
Bennett
A M
AM
Ho
A
A
Williamson
E D
ED
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D003001:Cloning, Molecular; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015010:Yersinia pestis / Q000276:immunology*
D000942:Antigens, Bacterial; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins