Bimolecular fluorescence complementation

Bimolecular fluorescence complementation (also known as BiFC) is a technology typically used to validate protein interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.

Biochemical complementation was first reported in subtilisin-cleaved bovine pancreatic ribonuclease, then expanded using β-galactosidase mutants that allowed cells to grow on lactose.

Recognition of many proteins' ability to spontaneously assemble into functional complexes as well as the ability of protein fragments to assemble as a consequence of the spontaneous functional complex assembly of interaction partners to which they are fused was later reported for ubiquitin fragments in yeast protein interactions.

In 2000, Ghosh et al developed a system that allowed a green fluorescent protein (GFP) to be reassembled using an anti-parallel leucine zipper in E. coli cells. This was achieved by dissecting GFP into C- and N-terminal GFP fragments. As the GFP fragment was attached to each leucine zipper by a linker, the heterodimerisation of the anti-parallel leucine zipper resulted in a reconstituted, or re-formed, GFP protein that could be visualised. The successful fluorescent signal indicated that the separate GFP peptide fragments were able to correctly reassemble and achieve tertiary folding. It was, therefore, postulated that using this technique, fragmented GFP could be used to study interaction of protein–protein pairs that have their N–C termini in close proximity.

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