Wikipedia

Protein-fragment complementation assay

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 edit

 
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:

Genome-wide applications edit

The methods mentioned above have been applied to whole genomes, e.g. yeast[3] or syphilis bacteria.[19]

References edit

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  9. ^ Tchekanda E, Sivanesan D, Michnick SW (June 2014). "An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions". Nature Methods. 11 (6): 641–4. doi:10.1038/nmeth.2934. PMID 24747815. S2CID 1958433.
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  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 (December 2014). "A versatile platform to analyze low-affinity and transient protein-protein interactions in living cells in real time". Cell Reports. 9 (5): 1946–58. doi:10.1016/j.celrep.2014.10.058. PMC 4269221. PMID 25464845.
  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 (December 2012). "Comparative analysis of virus-host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase". Methods. 58 (4): 349–59. doi:10.1016/j.ymeth.2012.07.029. PMC 3546263. PMID 22898364.
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  16. ^ Kolkhof P, Werthebach M, van de Venn A, Poschmann G, Chen L, Welte M, Stühler K, Beller M (March 2017). "A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions". Molecular & Cellular Proteomics. 16 (3): 329–345. doi:10.1074/mcp.M116.061499. PMC 5340998. PMID 27956707.
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  18. ^ 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. ISBN 978-1-61779-291-5. PMID 21938623.
  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. Bibcode:2008PLoSO...3.2292T. doi:10.1371/journal.pone.0002292. ISSN 1932-6203. PMC 2386257. PMID 18509523.

Further reading edit

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