219
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_14, © Springer Science+Business Media LLC 2017
Chapter 14
Characterizing Dynamic Protein–Protein Interactions Using
the Genetically Encoded Split Biosensor Assay Technique
Split TEV
Jan P. Wintgens, Moritz J. Rossner, and Michael C. Wehr
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 highthroughput 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.
Key words Protein–protein interaction, Split TEV, Biosensor, Split biosensor assay, RTK, GPCR,
Phosphorylation-dependent interactions, Dose-response assay, Compound profiling
1 Introduction
Studying protein–protein interactions (PPIs) is paramount to
understand cellular signaling and responses thereof. The majority
of PPIs are precisely regulated both in time and space, and mediate
distinct cellular activities, including differentiation, proliferation,
apoptosis, and inflammation. In terms of signaling, cell surface
receptors and highly interlinked intracellular proteins, or hubs, are
of special interest, as these proteins integrate key signaling activities
to regulate global cellular responses. For cytosolic proteins, pivotal
associations with a strong implication on downstream signaling are
1.1 Genetically
Encoded Biosensor
Assays for Studying
Protein–Protein
Interactions
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_14, © Springer Science+Business Media LLC 2017
Chapter 14
Characterizing Dynamic Protein–Protein Interactions Using
the Genetically Encoded Split Biosensor Assay Technique
Split TEV
Jan P. Wintgens, Moritz J. Rossner, and Michael C. Wehr
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 highthroughput 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.
Key words Protein–protein interaction, Split TEV, Biosensor, Split biosensor assay, RTK, GPCR,
Phosphorylation-dependent interactions, Dose-response assay, Compound profiling
1 Introduction
Studying protein–protein interactions (PPIs) is paramount to
understand cellular signaling and responses thereof. The majority
of PPIs are precisely regulated both in time and space, and mediate
distinct cellular activities, including differentiation, proliferation,
apoptosis, and inflammation. In terms of signaling, cell surface
receptors and highly interlinked intracellular proteins, or hubs, are
of special interest, as these proteins integrate key signaling activities
to regulate global cellular responses. For cytosolic proteins, pivotal
associations with a strong implication on downstream signaling are
1.1 Genetically
Encoded Biosensor
Assays for Studying
Protein–Protein
Interactions