3.1. Generation of BioID fusion protein

It is critical not to disrupt any known targeting motifs and post-translational modifications by the fusion of BioID. If GFP has been fused to the bait (BioID is about the size of GFP) and shown not to perturb targeting and/or function then this inform BioID placement (see Note 4).

Cloning a BioID-fusion protein typically requires PCR. Using recombinational cloning techniques, PCR products can be inserted to the BioID vectors without restriction enzyme digestions. This technique provides more freedom to choose restriction enzymes in the vector.

The biotinylation range can be modulated by flexible linkers (e.g. BioID-flexible linker-bait). The length of the flexible linker may vary depending on the goals of the experiment. Short flexible linkers may also relieve any steric hindrance between the bait and BioID ligase.

If ectopic expression of the fusion protein needs to be inducibly regulated due to unwanted biological effects, consider using an inducible expression system. The BioID system itself is functional without inducible expression since biotinylation is regulated by the addition of excess biotin.