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Home -> Cell Biology -> Cell-based Protein Protein Interaction Assays (targetSCREENER Assays)

Cell-based Protein Protein Interaction Assays (targetSCREENER Assays)

• Genetically encoded tools for cell-based high-throughput screening
• Monitor cellular signalling events in living cells, either at the membrane or in the cytosol.
• Premade kits available for various drug targets such as G protein-coupled receptors and receptor kinases
• Robust, sensitive, quantitative, and customizable assay format

targetSCREENER assays from our Partner SYSTASY are based on their proprietary splitSENSOR Technology. They are a highly sensitive, flexible and easy-to-use reporter assay system to monitor various cellular events in living cells, including the activity of drug targets. Various cellular events, such as regulated protein-protein interactions, can be robustly and quantitatively measured both at the membrane and in the cytosol of the cell. This feature makes it, for example, an invaluable tool to assess the activity of receptors as measured by the regulated binding to their cognate cytosolic adapters. Therefore, this technology can be applied in particular to assess compound actions on drug targets, such as G protein-coupled receptors and receptor tyrosine kinases.

As luciferase reporters are used as readout, the splitSENSOR technology is amenable to high-throughput applications including compound screening approaches. targetSCREENER assays are available for transient expression, and alternatively, assay components can be stably integrated into mammalian cells of choice using neomycin selection.

How it works

The splitSENSOR technology is based on the functional reconstitution of two previously inactive fragments derived from the non-native NIa protease of the tabacco etch virus (TEV protease) (Wehr et al., 2006). These fragments, either an N-terminal (NTEV) or C-terminal part (CTEV) of the TEV protease, are fused to interaction partners of choice. Upon interaction of the two candidate proteins, the NTEV and CTEV fragments get into close proximity, thereby regaining proteolytic activity. An active protease recognises and cleaves its target sequence, ENLYFQG, to release a previously inactive reporter molecule, i.e. an artificial transcriptional co-activator like Gal4-VP16 (GV). GV, in turn, translocates to the nucleus to activate a reporter gene, such as firefly luciferase (Fluc), producing light.

Image
splitSENSOR technology can be applied to monitor interactions at the membrane (left) and in the cytosol (right). For an efficient and robust assay involving membrane proteins or proteins that are localised to the sub-membrane compartment, the artificial transcriptional co-activator GV is fused to the NTEV moiety via a TEV protease cleavage site.


References:

The power and applicability of the splitSENSOR technology was demonstrated by various publications in high-class scientific journals:

[1] M. C. Wehr, R. Laage, U. Bolz, T. M. Fischer, S. Grünewald, S. Scheek, A. Bach, K.-A. Nave, and M. J. Rossner, ‘Monitoring regulated protein-protein interactions using split TEV’, Nat. Methods, vol. 3, no. 12, pp. 985–993, Dec. 2006.

[2] M. Wehr, L. Reinecke, A. Botvinnik, and M. Rossner, ‘Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV’, BMC Biotechnol., vol. 8, no. 1, p. 55, 2008.

[3] A. Genevet, M. C. Wehr, R. Brain, B. J. Thompson, and N. Tapon, ‘Kibra is a regulator of the Salvador/Warts/Hippo signaling network’, Dev. Cell, vol. 18, no. 2, pp. 300–308, Feb. 2010.

[4] A. Botvinnik, S. P. Wichert, T. M. Fischer, and M. J. Rossner, ‘Integrated analysis of receptor activation and downstream signaling with EXTassays’, Nat. Methods, vol. 7, no. 1, pp. 74–80, Jan. 2010.

[5] D. C. Gray, S. Mahrus, and J. A. Wells, ‘Activation of Specific Apoptotic Caspases with an Engineered Small-Molecule-Activated Protease’, Cell, vol. 142, no. 4, pp. 637–646, Aug. 2010.

[6] M. S. Djannatian, S. Galinski, T. M. Fischer, and M. J. Rossner, ‘Studying G protein-coupled receptor activation using split-tobacco etch virus assays’, Anal. Biochem., vol. 412, no. 2, pp. 141–152, May 2011.

[7] A. Botvinik and M. J. Rossner, ‘Linking cellular signalling to gene expression using EXT-encoded reporter libraries’, Methods Mol. Biol. Clifton NJ, vol. 786, pp. 151–166, 2012.

[8] A. Botvinik and M. J. Rossner, ‘Integrated measurement of split TEV and cis-regulatory assays using EXT encoded reporter libraries’, Methods Mol. Biol. Clifton NJ, vol. 812, pp. 309–323, 2012.

[9] X. Capdevila-Nortes, T. López-Hernández, F. Ciruela, and R. Estévez, ‘A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells’, Anal. Biochem., Jan. 2012.

[10] M. C. Wehr, M. V. Holder, I. Gailite, R. E. Saunders, T. M. Maile, E. Ciirdaeva, R. Instrell, M. Jiang, M. Howell, M. J. Rossner, and N. Tapon, ‘Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila’, Nat. Cell Biol., vol. 15, no. 1, pp. 61–71, Jan. 2013.


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