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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.〔Kerppola, T. K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Protoc. 1, 1278–1286 (2006).〕 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.〔Morell, M., Espargaro, A., Aviles, F. X. & Ventura, S. Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry. Nat. Protoc. 3, 22–33 (2008).〕 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. ==History== Biochemical complementation was first reported in subtilisin-cleaved bovine pancreatic ribonuclease, then expanded using β-galactosidase mutants that allowed cells to grow on lactose.〔Richards, F. M. On the Enzymic Activity of Subtilisin-Modified Ribonuclease. Proc. Natl. Acad. Sci. U. S. A. 44, 162–166 (1958).〕〔Ullmann, A., Jacob, F. & Monod, J. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24, 339–343 (1967).〕〔Ullmann, A., Jacob, F. & Monod, J. On the subunit structure of wild-type versus complemented beta-galactosidase of Escherichia coli. J. Mol. Biol. 32, 1–13 (1968).〕 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.〔Johnsson, N. & Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. U. S. A. 91, 10340-10344 (1994).〕 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.〔Ghosh, I., Hamilton, A. D. & Regan, L. Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. Journal of the American Chemical Society 122, 5658 (2000).〕 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. After the demonstration of successful fluorescent protein fragment reconstitution in mammalian cells, Hu ''et al''. described the use of fragmented yellow fluorescent protein (YFP) in the investigation of bZIP and Rel family transcription factor interactions.〔 This was the first report bZIP protein interaction regulation by regions outside of the bZIP domain, regulation of subnuclear localization of the bZIP domains Fos and Jun by their different interacting partners, and modulation of transcriptional activation of bZIP and Rel proteins through mutual interactions. In addition, this study was the first report of an ''in vivo'' technique, now known as the bimolecular fluorescence complementation (BiFC) assay, to provide insight into the structural basis of protein complex formation through detection of fluorescence caused by the assembly of fluorescent reporter protein fragments tethered to interacting proteins.〔Hu, C. D., Chinenov, Y. & Kerppola, T. K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798 (2002).〕 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Bimolecular fluorescence complementation」の詳細全文を読む スポンサード リンク
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