Abstract
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.
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Acknowledgements
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).
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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
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DOI: https://doi.org/10.1038/nprot.2006.14
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