Within the field of molecular biology, a protein-fragment complementation assay, or PCA, is a method for the identification and quantification of protein–protein interactions. In the PCA, the proteins of interest ("bait" and "prey") are each covalently linked to fragments of a third protein (e.g. DHFR, which acts as a "reporter"). Interaction between the bait and the prey proteins brings the fragments of the reporter protein in close proximity to allow them to form a functional reporter protein whose activity can be measured. This principle can be applied to many different reporter proteins and is also the basis for the yeast two-hybrid system, an archetypical PCA assay.
Split protein assays
General principle of the protein complementation assay: a protein is split into two (N- and C-terminal) halves and reconstituted by two interacting proteins that are fused to the N and C halves (here called "bait" and "prey" because a bait protein can be used to find an interacting prey protein). The activity of the reconstituted protein should be easily detectable, e.g. as in the green fluorescent protein (GFP).
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:
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]
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^Remy I, Ghaddar G, Michnick SW (2007). "Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions". Nature Protocols. 2 (9): 2302–6. doi:10.1038/nprot.2007.356. PMID17853887. S2CID7607566.
^Ma Y, Nagamune T, Kawahara M (September 2014). "Split focal adhesion kinase for probing protein–protein interactions". Biochemical Engineering Journal. 90: 272–278. Bibcode:2014BioEJ..90..272M. doi:10.1016/j.bej.2014.06.022.
^Barnard E, Timson DJ (2010). "Split-EGFP Screens for the Detection and Localisation of Protein–Protein Interactions in Living Yeast Cells". Molecular and Cell Biology Methods for Fungi. Methods in Molecular Biology. Vol. 638. pp. 303–17. doi:10.1007/978-1-60761-611-5_23. ISBN978-1-60761-610-8. PMID20238279.
^Blakeley BD, Chapman AM, McNaughton BR (August 2012). "Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo". Molecular BioSystems. 8 (8): 2036–40. doi:10.1039/c2mb25130b. PMID22692102.
^Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, Rossner MJ (December 2006). "Monitoring regulated protein-protein interactions using split TEV". Nature Methods. 3 (12): 985–93. doi:10.1038/nmeth967. PMID17072307. S2CID37120401.
^Dünkler A, Müller J, Johnsson N (2012). "Detecting Protein–Protein Interactions with the Split-Ubiquitin Sensor". Gene Regulatory Networks. Methods in Molecular Biology. Vol. 786. pp. 115–30. doi:10.1007/978-1-61779-292-2_7. ISBN978-1-61779-291-5. PMID21938623.
