Characterizing Dynamic Protein-Protein Interactions Using the Genetically Encoded Split Biosensor Assay Technique Split TEV

Methods Mol Biol. 2017;1596:219-238. doi: 10.1007/978-1-4939-6940-1_14.

Abstract

Dynamic protein-protein interactions (PPIs) are fundamental building blocks of cellular signaling and monitoring their regulation promotes the understanding of signaling in health and disease. Genetically encoded split protein biosensor assays, such as the split TEV method, have proved to be highly valuable when studying regulated PPIs in living cells. Split TEV is based on the functional complementation of two previously inactive TEV protease fragments fused to interacting proteins and provides a robust, sensitive and flexible readout to monitor PPIs both at the membrane and in the cytosol. Thus, split TEV can be used to analyze interactomes of receptors, membrane-associated proteins, and cytosolic proteins. In particular, split TEV is useful to assay activities of relevant drug targets, such as receptor tyrosine kinases and G protein-coupled receptors, in compound screens. As split TEV uses genetically encoded readouts, including standard reporters based on fluorescence and luminescence, the technique can also be combined with scalable molecular barcode reporter systems, allowing the integration into multiplexed high-throughput assay approaches. Split TEV can be used in standard heterologous cell lines and primary cell types, including neurons, either in a transient or stably integrated format. When using cell lines, the basic protocol takes 30-96 h to complete, depending on the complexity of the experimental question addressed.

Keywords: Biosensor; Compound profiling; Dose-response assay; GPCR; Phosphorylation-dependent interactions; Protein–protein interaction; RTK; Split TEV; Split biosensor assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay / methods
  • Biosensing Techniques
  • Cell Line
  • Cell Line, Tumor
  • Endopeptidases / genetics*
  • HEK293 Cells
  • Humans
  • Membrane Proteins / genetics
  • PC12 Cells
  • Protein Interaction Mapping / methods
  • Rats
  • Receptors, G-Protein-Coupled / genetics
  • Signal Transduction / genetics

Substances

  • Membrane Proteins
  • Receptors, G-Protein-Coupled
  • Endopeptidases
  • TEV protease