Reverse mammalian protein-protein interaction trap (MAPPIT) is a mammalian reverse two-hybrid technology. The method is adapted from the forward MAPPIT technique, a two-hybrid complementation system in which the interaction between a bait-fusion protein and a prey-fusion protein restores ligand-dependent cytokine receptor signaling. In the reverse mode described in detail here, a positive readout is generated on disruption of the designated protein-protein interactions. Reverse MAPPIT functions in intact human cells, facilitating simultaneous analysis of disruption, toxicity and permeability of the tested compounds, making it particularly suitable for screening for molecules that target therapeutically interesting protein-protein interactions or for mapping the interaction interface between proteins. The total handling time of a typical reverse MAPPIT experiment is ∼9 h and is spread over 4–5 d.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Arkin, M.R. & Wells, J.A. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat. Rev. Drug Discov. 3, 301–317 (2004).
Vidal, M., Braun, P., Chen, E., Boeke, J.D. & Harlow E. Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proc. Natl. Acad. Sci. USA 93, 10321–10326 (1996).
Eyckerman, S. et al. Design and application of a cytokine-receptor-based interaction trap. Nat. Cell Biol. 3, 1114–1119 (2001).
Cook, W.S. & Unger, R.H. Protein tyrosine phosphatase 1B: a potential leptin resistance factor of obesity. Dev. Cell 2, 385–387 (2002).
Eyckerman, S. et al. Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells. Nat. Methods 2, 427–432 (2005).
Milstein, S. & Vidal, M. Perturbing interactions. Nat. Methods 2, 412–414 (2005).
Takebe, Y. et al. SRα promoter: an efficient and versatile mammalian cDNA expression system composed of the Simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell Biol. 8, 466–472 (1988).
Ausubel, F.M. et al. (eds.) in Current Protocols in Molecular Biology Suppl. 56 15.1.1–15.1.14 (Wiley Publishing, Indianapolis, IN, 2005).
Eyckerman, S. et al. Design and use of a mammalian protein-protein interaction trap (MAPPIT). Sci. STKE 162, PL18 (2002).
We thank Prof. Dr. J. Vandekerckhove for continued support and Dominiek Catteeuw and Annick Verhee for technical assistance in the development of the reverse MAPPIT method. This project was supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen; GBOU 010090), Ghent University (UGent; GOA 12051401) and the valorization fund of the Flanders Interuniversity Institute for Biotechnology (VIB). I.L. and S.E. are Postdoctoral Fellows of the Fund for Scientific Research–Flanders (FWO-V).
The authors declare no competing financial interests.
About this article
Cite this article
Lemmens, I., Lievens, S., Eyckerman, S. et al. Reverse MAPPIT detects disruptors of protein-protein interactions in human cells. Nat Protoc 1, 92–97 (2006). https://doi.org/10.1038/nprot.2006.14
A Chemically Inducible Helper Module for Detecting Protein–Protein Interactions with Tunable Sensitivity Based on KIPPIS
Analytical Chemistry (2017)
Nature Chemical Biology (2015)
Interactome mapping for analysis of complex phenotypes: Insights from benchmarking binary interaction assays