High-throughput strategy for identification of Mycobacterium tuberculosis membrane protein expression conditions using folding reporter GFP.

Mycobacterium tuberculosis membrane protein biochemistry and structural biology studies are often hampered by challenges in protein expression and selection for well-expressing protein candidates, suitable for further investigation. Here we present a folding reporter GFP (frGFP) assay, adapted for M. tuberculosis membrane protein screening in Escherichia coli Rosetta 2 (DE3) and Mycobacterium smegmatis mc2 4517. This method allows protein expression condition screening for multiple protein targets simultaneously by monitoring frGFP fluorescence in growing cells. We discuss the impact of common protein expression conditions on 42 essential M. tuberculosis H37Rv helical transmembrane proteins and establish the grounds for their further analysis. We have found that the basal expression of the lac operon in the T7-promoter expression system generally leads to high recombinant protein yield in M. smegmatis, and we suggest that a screening condition without the inducer is included in routine protein expression tests. In addition to the general observations, we describe conditions allowing high-level expression of more than 25 essential M. tuberculosis membrane proteins, containing 2 to 13 transmembrane helices. We hope that these findings will stimulate M. tuberculosis membrane protein research and aid the efforts in drug development against tuberculosis.