Protein-fragment complementation assay
Within the field of
Split protein assays
Any protein that can be split into two parts and reconstituted non-covalently to form a functional protein may be used in a PCA. The two fragments however have low affinity for each other and must be brought together by other interacting proteins fused to them (often called "bait" and "prey" since the bait protein can be used to identify a prey protein, see figure). The protein that produces a detectable readout is called "reporter". Usually enzymes which confer resistance to nutrient deprivation or antibiotics, such as dihydrofolate reductase or beta-lactamase respectively, or proteins that give colorimetric or fluorescent signals are used as reporters. When fluorescent proteins are reconstituted the PCA is called Bimolecular fluorescence complementation assay. The following proteins have been used in split protein PCAs:
- Beta-lactamase[1][2]
- Dihydrofolate reductase (DHFR)[3]
- Focal adhesion kinase (FAK)[4]
- Gal4, a yeast transcription factor (as in the classical yeast two-hybrid system)
- GFP (split-GFP), e.g. EGFP (enhanced green fluorescent protein)[5][6][7]
- Horseradish peroxidase[8]
- Infrared fluorescent protein IFP1.4, an engineered chromophore-binding domain (CBD) of a bacteriophytochrome from Deinococcus radiodurans [9]
- LacZ (beta-galactosidase)[10]
- Luciferase,[11][12] including ReBiL (recombinase enhanced bimolecular luciferase)[13] and Gaussia princeps luciferase.[14] Commercial products using luciferase include NanoLuc and NanoBIT.[15] A modification has also been developed for lipid droplet-associated interactions.[16]
- TEV (Tobacco etch virus protease) [17]
- Ubiquitin[18]
Genome-wide applications
The methods mentioned above have been applied to whole genomes, e.g. yeast[3] or syphilis bacteria.[19]
References
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- .
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- ^ Fujikawa, Y. et al. (2014) Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format. Rice 7:11
- PMID 25464845.
- PMID 22898364.
- ^ Binkowski B, Eggers C, Butler B, Schwinn M, Slater M, Machleidt T, Cong M, Wood K, Fan F (May 2016). "Monitoring intracellular protein interactions using NanoLuc® Binary Technology (NanoBiTTM)" (PDF). Promega.
- PMID 27956707.
- S2CID 37120401.
- PMID 21938623.
- PMID 18509523.
Further reading
- Rochette S, Diss G, Filteau M, Leducq JB, Dubé AK, Landry CR (March 2015). "Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells". Journal of Visualized Experiments (97). PMID 25867901.