Protein-fragment complementation assay explained

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

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 or syphilis bacteria.^[19]

Further reading

• Rochette S, Diss G, Filteau M, Leducq JB, Dubé AK, Landry CR . Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells . Journal of Visualized Experiments . 97 . March 2015 . 25867901 . 4401175 . 10.3791/52255 .

Notes and References

1. Park JH, Back JH, Hahm SH, Shim HY, Park MJ, Ko SI, Han YS . Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs . Journal of Microbiology and Biotechnology . 17 . 10 . 1607–15 . October 2007 . 18156775 .
2. Remy I, Ghaddar G, Michnick SW . Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions . Nature Protocols . 2 . 9 . 2302–6 . 2007 . 17853887 . 10.1038/nprot.2007.356 . 7607566 .
3. Tarassov K, Messier V, Landry CR, Radinovic S, Serna Molina MM, Shames I, Malitskaya Y, Vogel J, Bussey H, Michnick SW . An in vivo map of the yeast protein interactome . Science . 320 . 5882 . 1465–70 . June 2008 . 18467557 . 10.1126/science.1153878 . 2008Sci...320.1465T . 1732896 .
4. Ma . Yidan . Nagamune . Teruyuki . Kawahara . Masahiro . vanc . Split focal adhesion kinase for probing protein–protein interactions . Biochemical Engineering Journal . September 2014 . 90 . 272–278 . 10.1016/j.bej.2014.06.022 . 2014BioEJ..90..272M .
5. Book: Barnard E, Timson DJ . Split-EGFP Screens for the Detection and Localisation of Protein–Protein Interactions in Living Yeast Cells . Molecular and Cell Biology Methods for Fungi . 638 . 303–17 . 2010 . 20238279 . 10.1007/978-1-60761-611-5_23 . 978-1-60761-610-8 . Methods in Molecular Biology .
6. Blakeley BD, Chapman AM, McNaughton BR . Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo . Molecular BioSystems . 8 . 8 . 2036–40 . August 2012 . 22692102 . 10.1039/c2mb25130b .
7. Cabantous S, Nguyen HB, Pedelacq JD, Koraïchi F, Chaudhary A, Ganguly K, Lockard MA, Favre G, Terwilliger TC, Waldo GS . A new protein-protein interaction sensor based on tripartite split-GFP association . Scientific Reports . 3 . 2854 . October 2013 . 24092409 . 3790201 . 10.1038/srep02854 . 2013NatSR...3E2854C .
8. Martell JD, Yamagata M, Deerinck TJ, Phan S, Kwa CG, Ellisman MH, Sanes JR, Ting AY . A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses . Nature Biotechnology . 34 . 7 . 774–80 . July 2016 . 27240195 . 4942342 . 10.1038/nbt.3563 .
9. Tchekanda E, Sivanesan D, Michnick SW . An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions . Nature Methods . 11 . 6 . 641–4 . June 2014 . 24747815 . 10.1038/nmeth.2934 . 1958433 .
10. Rossi F, Charlton CA, Blau HM . Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation . Proceedings of the National Academy of Sciences of the United States of America . 94 . 16 . 8405–10 . August 1997 . 9237989 . 22934 . 10.1073/pnas.94.16.8405 . 1997PNAS...94.8405R . free .
11. Cassonnet P, Rolloy C, Neveu G, Vidalain PO, Chantier T, Pellet J, Jones L, Muller M, Demeret C, Gaud G, Vuillier F, Lotteau V, Tangy F, Favre M, Jacob Y . Benchmarking a luciferase complementation assay for detecting protein complexes . Nature Methods . 8 . 12 . 990–2 . November 2011 . 22127214 . 10.1038/nmeth.1773 . 9377872 .
12. 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
13. Li YC, Rodewald LW, Hoppmann C, Wong ET, Lebreton S, Safar P, Patek M, Wang L, Wertman KF, Wahl GM . A versatile platform to analyze low-affinity and transient protein-protein interactions in living cells in real time . Cell Reports . 9 . 5 . 1946–58 . December 2014 . 25464845 . 4269221 . 10.1016/j.celrep.2014.10.058 .
14. Neveu G, Cassonnet P, Vidalain PO, Rolloy C, Mendoza J, Jones L, Tangy F, Muller M, Demeret C, Tafforeau L, Lotteau V, Rabourdin-Combe C, Travé G, Dricot A, Hill DE, Vidal M, Favre M, Jacob Y . Comparative analysis of virus-host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase . Methods . 58 . 4 . 349–59 . December 2012 . 22898364 . 3546263 . 10.1016/j.ymeth.2012.07.029 .
15. Web site: Brock . Binkowski . Christopher . Eggers . Braeden . Butler . Marie . Schwinn . Michael . Slater . Thomas . Machleidt . Mei . Cong . Keith . Wood . Frank . Fan . vanc . Monitoring intracellular protein interactions using NanoLuc® Binary Technology (NanoBiTTM) . May 2016 . Promega .
16. Kolkhof P, Werthebach M, van de Venn A, Poschmann G, Chen L, Welte M, Stühler K, Beller M . A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions . Molecular & Cellular Proteomics . 16 . 3 . 329–345 . March 2017 . 27956707 . 5340998 . 10.1074/mcp.M116.061499 . free .
17. Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, Rossner MJ . Monitoring regulated protein-protein interactions using split TEV . Nature Methods . 3 . 12 . 985–93 . December 2006 . 17072307 . 10.1038/nmeth967 . 37120401 .
18. Book: Dünkler A, Müller J, Johnsson N . Detecting Protein–Protein Interactions with the Split-Ubiquitin Sensor . Gene Regulatory Networks . 786 . 115–30 . 2012 . 21938623 . 10.1007/978-1-61779-292-2_7 . 978-1-61779-291-5 . Methods in Molecular Biology .
19. Titz. Björn. Rajagopala. Seesandra V.. Goll. Johannes. Häuser. Roman. McKevitt. Matthew T.. Palzkill. Timothy. Uetz. Peter. 2008-05-28. The binary protein interactome of Treponema pallidum--the syphilis spirochete. PLOS ONE. 3. 5. e2292. 10.1371/journal.pone.0002292. 1932-6203. 2386257. 18509523. 2008PLoSO...3.2292T. free.