Recombinant production of Mtb antigens and their purification by affinity chromatography

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 HisTrap^TMFF 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.