Schematic of the strategy used to purify protein complexes and identify protein–protein interactions in mycobacteria.

<p>(A) For expression of selected bait, pKW08-derived plasmids were engineered. Genes selected for further tests were cloned into the constructed vectors, allowing fusion with specific tags. (B) To minimize differences between tags, all tags were designed in a similar manner, containing the SH-quant, a cleavage site for TEV protease, a spacer and a single specific epitope terminating with a stop codon. Recombinant proteins were expressed in mycobacterial cells and the bait was purified on an epitope-specific resin. (C) Anti-GFP nanobodies prepared for this work were immobilized on activated Sepharose beads, and eGFP binding was examined by microscopy. (D) The overview of the purification procedure followed by LC–MS/MS and protein identification by MaxQuant software. (E) eGFP can be used to visualize the subcellular localization of a target protein.