Protein–protein interaction screening

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The screening of protein–protein interactions refers to the identification of protein interactions with high-throughput screening methods such as computer- and/or robot-assisted plate reading, flow cytometry analyzing.

The interactions between proteins are central to virtually every process in a living cell. Information about these interactions improves understanding of diseases and can provide the basis for new therapeutic approaches.

Methods to screen protein–protein interactions[edit]

Though there are many methods to detect protein–protein interactions, the majority of these methods—such as Co-immunoprecipitation, Fluorescence resonance energy transfer (FRET) and dual polarisation interferometry—are not screening approaches.

Ex vivo or in vivo methods[edit]

Methods that screen protein–protein interactions in the living cells.

  • Bimolecular Fluorescence Complementation (BiFC) is a new technique for observing the interactions of proteins. Combining it with other new techniques DERB can enable the screening of protein–protein interactions and their modulators.[1]
  • The yeast two-hybrid screen investigates the interaction between artificial fusion proteins inside the nucleus of yeast. This approach can identify the binding partners of a protein without bias. However, the method has a notoriously high false-positive rate, which makes it necessary to verify the identified interactions by co-immunoprecipitation.[2]

In-vitro methods[edit]

  • The Tandem affinity purification (TAP) method allows the high-throughput identification of proteins interactions. In contrast with the Y2H approach, the accuracy of the method can be compared to those of small-scale experiments (Collins et al., 2007) and the interactions are detected within the correct cellular environment as by co-immunoprecipitation. However, the TAP tag method requires two successive steps of protein purification, and thus can not readily detect transient protein–protein interactions. Recent genome-wide TAP experiments were performed by Krogan et al., 2006,[3] and Gavin et al., 2006,[4] providing updated protein interaction data for yeast organisms.
  • Chemical crosslinking is often used to "fix" protein interactions in place before trying to isolate/identify interacting proteins. Common crosslinkers for this application include the non-cleavable [NHS-ester] crosslinker, [bis-sulfosuccinimidyl suberate] (BS3); a cleavable version of BS3, [dithiobis(sulfosuccinimidyl propionate)](DTSSP); and the [imidoester] crosslinker [dimethyl dithiobispropionimidate] (DTBP) that is popular for fixing interactions in ChIP assays.[5]

See also[edit]

References[edit]

  1. ^ Lu JP, Beatty LK, Pinthus JH (2008). "Dual expression recombinase based (DERB) single vector system for high throughput screening and verification of protein interactions in living cells". Nature Precedings. hdl:10101/npre.2008.1550.2. 
  2. ^ Fields S (2005). "High-throughput two-hybrid analysis: The promise and the peril". FEBS Journal 272 (21): 5391–5399. doi:10.1111/j.1742-4658.2005.04973.x. PMID 16262681. 
  3. ^ Krogan, Nevan J.; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P.; Punna, Thanuja; Peregrín-Alvarez, José M.; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D.; Paccanaro, Alberto; Bray, James E.; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P.; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M.; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R.; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J.; Gandi, Kiran; Thompson, Natalie J.; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H. Y.; Butland, Gareth; Altaf-Ul, Amin M.; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S.; Ingles, C. James; Hughes, Timothy R.; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J.; Emili, Andrew; Greenblatt, Jack F. (21 March 2006). "Global landscape of protein complexes in the yeast Saccharomyces cerevisiae". Nature 440 (7084): 637–643. doi:10.1038/nature04670. PMID 16554755. 
  4. ^ Gavin, Anne-Claude; Aloy, Patrick; Grandi, Paola; Krause, Roland; Boesche, Markus; Marzioch, Martina; Rau, Christina; Jensen, Lars Juhl; Bastuck, Sonja; Dümpelfeld, Birgit; Edelmann, Angela; Heurtier, Marie-Anne; Hoffman, Verena; Hoefert, Christian; Klein, Karin; Hudak, Manuela; Michon, Anne-Marie; Schelder, Malgorzata; Schirle, Markus; Remor, Marita; Rudi, Tatjana; Hooper, Sean; Bauer, Andreas; Bouwmeester, Tewis; Casari, Georg; Drewes, Gerard; Neubauer, Gitte; Rick, Jens M.; Kuster, Bernhard; Bork, Peer; Russell, Robert B.; Superti-Furga, Giulio (21 January 2006). "Proteome survey reveals modularity of the yeast cell machinery". Nature 440 (7084): 631–636. doi:10.1038/nature04532. PMID 16429126. 
  5. ^ Chen CS, Zhu H (2006). "Protein microarrays". Biotechniques 40 (4): 423, 425, 427. doi:10.2144/06404TE01. PMID 16629388. 

External links[edit]

Protein–protein interaction databases[edit]