Expression, purification and initial characterization of LysE membrane exporter from Mycobacterium tuberculosis: Towards comprehensive functional and structural study

Abstract

Mycobacterium tuberculosis (Mtb) causes the very severe disease tuberculosis. To fight Mtb infection, detailed understanding of the pathogen molecular mechanisms in the host is needed. To this end, comprehensive knowledge about Mtb membrane transport system, which is currently not well researched, is a high priority. Our focus is on the Mtb‐encoded L‐lysine exporter (LysE), which removes the excess of metabolically produced basic amino acids from the bacterial cytosol. LysE is a target for inhibition, since its deficiency leads to toxic levels of L‐lysine and suppression of bacterial growth. The most studied H37Rv strain of Mtb encodes two LysEs, but one of then, the Rv1986 protein, is considered to be the primary exporter. Also, Rv1986 expression is increased during the early stages of Mtb infection under conditions of hypoxia, thus, the protein could be a vaccine target. Therefore, we selected to study this protein. To do so, we developed custom protocols for its cloning, expression, purification, and initial characterization. To overcome the low expression levels in E. coli, we fused LysE at its C‐terminus to a combination of FLAG and 8xHistidine tags. Through these tags, we employed a dual‐affinity purification. We purified the protein in lipodiscs made of native E. coli membranes and in detergent. We obtained highly pure protein as confirmed by SDS‐PAGE and Western blotting. Both techniques visualized multiple bands at LysE suggesting protein oligomerization. We further created and purified a single cysteine mutant N175C for spin labeling. Pulse EPR study on it also points to multimeric protein organization. These studies as well as electron microscopy on LysE‐nanogold particle complexes are ongoing. To the best of our knowledge, this is the first highly‐pure LysE produced, which will facilitate further thorough investigations of the functional mechanisms of this protein.Support or Funding InformationNIAID R03 AI137735