DefinePK

Abstract

Effective control of TB, the second largest cause of human deaths, is based on early diagnosis, proper treatment with availability of an efficacious vaccine. The antigen-based detection of antibodies and formulation of subunit anti-TB vaccines require highly purified immunogenic Mtb antigens. In this study three immunogenic Mtb antigens EspC, HspX and TB10.4 were cloned and expressed in E. coli through recombinant DNA techniques. SDS-PAGE was performed to check molecular sizes of EspC (13kDa), HspX (18kDa) and TB10.4 (11kDa) proteins. After expression, all the polyhistidine-tagged (6x-His) proteins were purified through an automated AKTA purifier FPLC system using HisTrapTMFF column. This purification system separates the proteins of interest through a continuous rather than step-wise imidazole gradient. The fractions containing the required protein were pooled each of the EspC, HspX and TB10.4 preparations thus obtained were more than 90% purified. The percentage recoveries of HspX, EspC, TB10.4 proteins were 12.0, 5.5 and 7.1, respectively. These preparations should be suitable for validation through immunological studies.