Production and affinity purification of diverse recombinant Mtb antigens

Abstract

Tuberculosis (TB) remains a global health threat, and effective disease control depends on timely diagnosis alongside appropriate treatment, while a robust vaccine could further reduce disease incidence. For both serological diagnosis and vaccine development, highly purified Mtb (Mycobacterium tuberculosis) antigens with strong immunogenicity are essential. In this study, we aimed to clone, express, and purify three Mtb recombinant proteins — Hrp1 (15.5kDa), HspX (16kDa), and Cfp17 (22kDa) — in soluble form using Escherichia coli. The target genes were amplified by PCR and inserted into the pET28a(+) vector. Recombinant proteins, carrying an N-terminal 6×His tag, were expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells under IPTG induction. Purification was carried out by Ni-NTA affinity chromatography. Densitometric analysis indicated expression levels of approximately 32% for Hrp1, 25% for HspX, and 16% for Cfp17. Final purified products exceeded 90% purity, with recoveries of 38.3% (Hrp1), 73% (HspX), and 45% (Cfp17). The recombinant Hrp1, HspX, and Cfp17 proteins, obtained with high purity, are therefore well suited for further evaluation in TB serodiagnostic assays and subunit-vaccine development.