BiFC & BiLC

Bimolecular fluorescence complementation (BiFC) is a technique that relies on the facilitated complementation of split fragments of a fluorescent protein. These fragments, on their own, lack any fluorescent properties. However, when brought close together, they regain their fluorescence. If BiFC works, it provides a powerful way to visualize dimeric proteins in live cells with exceptional spatial resolution.However, like many seemingly perfect solutions, BiFC faces some limitations. Firstly, it lacks a good temporal resolution due to the nature of the process. When the fragments are brought together, they need to undergo a series of steps, including complementing, connecting, and refolding, before they can regain their fluorescence. Secondly, a crucial drawback is the formation of a “covalent bond” between the fragments. It is believed that a bond is formed, effectively gluing the protein of interest together. Unfortunately, this bond is not ideally “dynamic,” which can hinder the ability to capture live cellular events.

Bimolecular luminescence complementation (BiLC) is quite similar to the BiFC technique, except that it uses split luciferases instead of split fluorescent proteins. Renilla luciferase (RLuc), a 314-amino acid protein, can be splitted from position 229. When these two RLuc fragments are brought close together, they refold and regain their luminescence properties. Recently, NanoLuc, a 171-amino acid protein, was also successfully splitted into two fragments (SmBit and LgBit), allowing us to use only a small fragment for protein tagging. Moreover, the complementation of luciferases is believed to be reversible, suggesting that a covalent bond is not necessary for regaining its luminescence properties. The refolding process is thought to be rapid, but unfortunately, we lack precise information about its speed.