N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (DapE) a Potential Biomarker of Mycobacterium Tuberculosis: Tag-Free Purification by Metal Affinity Chromatography: Tag-free purification of DapE by metal affinity chromatography

Abstract

DapE is an enzyme which is highly essential in lysine biosynthetic pathway for the growth and survival of Mycobacterium tuberculosis (Mtb) and other bacterial species although absent in human host. However, the sequence identity of Mtb-DapE with other mycobacterium and bacterial species estimated between 93 to 75% and approximately 50%, respectively. DapE is a metallo-enzyme requires few of transition metals for activity and bacterial proliferation. The homology model based structural studies including published structures revealed an availability of zinc interacting conserved amino residues in Mtb-DapE. In this study, we purified full length recombinant Mtb-DapE as tag-free (Mtb-DapETagFree) protein from inclusion bodies using zinc-NTA column. The single step purified protein observed with 96-98% purity and high yield. The indirect ELISA had 40% sensitivity using Mtb-DapE as an antigen against bovine tuberculosis (bTB) serum samples. IFA analysis with clear fluorescent spots of Mtb native DapE antigen in (3+) human TB positive sputum samples and recombinant Mtb-DapE positive control against Mtb-DapE polyclonal antibody are highly encouraging. The ELISA and IFA results incite for the consideration of Mtb-DapE in future development of quick and ideal TB detection assay along with other mycobacterium antigens. The future advanced detailed structural and functional studies using this highly purified and tag-free Mtb-DapE may provide discovery of antitubercular drug(s) and promising inhibitory molecules. HIGHLIGHTS Expression and purification of recombinant Mtb-DapETagFree. On-column refolded protein with 98% purity from inclusion bodies purified by zinc-NTA column affinity chromatography. The purified protein observed no aggregation and degradation even after 8 months storage. ELISA and IFA results supports for development of detection assay for TB from tubercular samples