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Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_11, © Springer Science+Business Media LLC 2017
Chapter 11
Protein and Protease Sensing by Allosteric Derepression
Hui Chin Goh, Farid J. Ghadessy, and Saurabh Nirantar
Abstract
Peptide motifs are crucial mediators of protein-protein interactions as well as sites of specific protease activity.
The detection and characterization of these events is therefore indispensable for a detailed understanding
of cellular regulation. Here, we present versatile and modular sensors that allow the user to detect protease
activity and protein-peptide interactions, as well as to screen for inhibitors using chromogenic, fluorescent,
or luminescent output.
Key words Protease sensors, Protein-protein interaction, Drug screening
1 Introduction
Sensing the binding of macromolecules to and cleavage of linear
peptide motifs is of great importance in elucidating the many regulatory processes in the cell. Several interaction-mediating domains
like the PDZ or SH2 families bind their cognate peptide to enable
colocalization of interacting proteins [1–3]. Similarly, processes as
diverse as viral maturation and blood clotting depend on the recognition of a specific peptide sequence by their target proteases
such as thrombin or tobacco etch virus (TEV) protease [4, 5].
In vitro studies with such peptides are very useful in detecting
and characterizing these functions. Typically, in vitro characterization of such events relies on techniques such as fluorescence polarization (FP) [6], enzyme-linked immunosorbent assays (ELISA)
[7], surface plasmon resonance (SPR) [8], and Forster resonance
energy transfer (FRET) [9]. While powerful, these methods require
substantial instrumentation and preparation and suffer from a number of limitations. These include interference from autofluorescent
molecules in case of FP measurements, multiple washing and
incubation steps in case of ELISAs, or low throughput for SPR.
Protein engineering-based methods have been developed to
address this need. These typically involve the insertion of a relevant
peptide or protein domain into a read-out capable enzyme such
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_11, © Springer Science+Business Media LLC 2017
Chapter 11
Protein and Protease Sensing by Allosteric Derepression
Hui Chin Goh, Farid J. Ghadessy, and Saurabh Nirantar
Abstract
Peptide motifs are crucial mediators of protein-protein interactions as well as sites of specific protease activity.
The detection and characterization of these events is therefore indispensable for a detailed understanding
of cellular regulation. Here, we present versatile and modular sensors that allow the user to detect protease
activity and protein-peptide interactions, as well as to screen for inhibitors using chromogenic, fluorescent,
or luminescent output.
Key words Protease sensors, Protein-protein interaction, Drug screening
1 Introduction
Sensing the binding of macromolecules to and cleavage of linear
peptide motifs is of great importance in elucidating the many regulatory processes in the cell. Several interaction-mediating domains
like the PDZ or SH2 families bind their cognate peptide to enable
colocalization of interacting proteins [1–3]. Similarly, processes as
diverse as viral maturation and blood clotting depend on the recognition of a specific peptide sequence by their target proteases
such as thrombin or tobacco etch virus (TEV) protease [4, 5].
In vitro studies with such peptides are very useful in detecting
and characterizing these functions. Typically, in vitro characterization of such events relies on techniques such as fluorescence polarization (FP) [6], enzyme-linked immunosorbent assays (ELISA)
[7], surface plasmon resonance (SPR) [8], and Forster resonance
energy transfer (FRET) [9]. While powerful, these methods require
substantial instrumentation and preparation and suffer from a number of limitations. These include interference from autofluorescent
molecules in case of FP measurements, multiple washing and
incubation steps in case of ELISAs, or low throughput for SPR.
Protein engineering-based methods have been developed to
address this need. These typically involve the insertion of a relevant
peptide or protein domain into a read-out capable enzyme such