Production of ABC Transporters from Mycobacterium tuberculosis for Functional and Structural Studies - A Platform for Membrane Protein Production

Abstract

Membrane proteins are considered the new challenge of Structural Biology given their medical relevance. More than 50% of the drug targets in industry involve membrane proteins, which represent about 30% of the known genomes. To set up in our laboratory a platform for production of membrane proteins for functional and structural studies, we have chosen as targets ABC transporters of Mycobacterium tuberculosis. ABC transporters are responsible for the multiple drug-resistance (MDR) in cancer cells and bacterial resistance, including in tuberculosis. ABC transporters in M. tuberculosis are poorly studied and the possibility of characterization of these proteins in this bacterium as well as defining the range of substrates, is a step toward developing new ways of controlling the disease. In this sense, it is intended to express, purify and produce those transporters in reconstituted membranes for functional studies of interaction with different substrates, function determination and structural analysis. To carry out this project, we established a collaboration with researchers Dr. Isabel de Moraes, head of the Membrane Protein Laboratory at the Diamond Light Source and Dr. Louise Bird of Oxford Protein Production Facility (OPPF) in Didcot, England. This collaboration seeks to initial cloning and expression analysis of large scale all carriers to identify the most promising targets. Preliminary results show that all the 17 chosen transporters of M. tuberculosis were cloned into expression vectors, and three were successfully expressed in sufficient quantities for the initial studies (two exporting drugs: Rv1819c and Rv1747 and a carrier glutamine Rv2563/2564). The optimization of conditions for expression of these transporters as well as methods of purification and cloning of the other sub-carriers with other approaches are currently underway.