Using BioID for the Identification of Interacting and Proximal Proteins in Subcellular Compartments in Toxoplasma gondii.

BioID is an in vivo biotinylation system developed to examine the proximal and interacting proteins of a bait protein within a subcellular compartment. This approach has been exploited in Toxoplasma for protein-protein interaction studies and proteomic characterizations of intracellular compartments. The BioID method requires constructing a translational fusion between a protein of interest and the promiscuous biotin ligase BirA∗ (a mutant of the E. coli protein BirA) which enables trafficking of the protein to the correct intracellular compartment and association with its partners. Proximity labelling occurs upon addition of biotin to the media and the biotinylated target proteins are then be purified using stringent conditions via streptavidin chromatography. In this chapter, we describe the methodology to fuse BirA∗ (or the newer variant BioID2) to a bait protein using endogenous gene tagging in Toxoplasma and then identify the proximal and interacting proteins using in vivo biotinylation, streptavidin purification and mass spectrometric analysis.