101
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_7, © Springer Science+Business Media LLC 2017
Chapter 7
Rational Design and Applications of Semisynthetic
Modular Biosensors: SNIFITs and LUCIDs
Helen Farrants, Julien Hiblot, Rudolf Griss, and Kai Johnsson
Abstract
Biosensors are used in many fields to measure the concentration of analytes, both in a cellular context and
in human samples for medical care. Here, we outline the design of two types of modular biosensors:
SNAP-tag-based indicators with a Fluorescent Intramolecular Tether (SNIFITs) and LUCiferase-based
Indicators of Drugs (LUCIDs). These semisynthetic biosensors quantitatively measure analyte concentrations in vitro and on cell surfaces by an intramolecular competitive mechanism. We provide an overview of
how to design and apply SNIFITs and LUCIDs.
Key words Biosensors, Protein engineering, Protein switches, Intramolecular ligands, Self-labeling
proteins, SNAP-tag, CLIP-tag, Polyproline linkers, Therapeutic drug monitoring, Cell-surface
biosensors
1 Introduction
Small molecule analytes are central to many biological systems.
The ability to quantify such analytes is important in basic research
to better understand anabolic, metabolic, and signal relay processes. It is important to quantify small molecules not only in basic
research, but also in healthcare, where it is crucial to be able to
monitor the levels of drugs and metabolites for therapeutic purposes. The use of biosensors is one method for this quantification,
and has many different applications [1, 2].
Several natural proteins bind small molecule analytes. If such a
binding protein undergoes a conformational change upon analyte
binding, it can be used as part of a biosensor system [3, 4]. For
example, it can be sandwiched in between two fluorescent proteins
that are Förster resonance energy transfer (FRET)-partners [5, 6]
or between two bioluminescent resonance energy transfer (BRET)partners [7]. In both cases, the change in distance and orientation
between the two resonance energy transfer (RET) partners is translated into changes in the optical properties of the system, which
1.1 SNIFITS
and LUCIDs:
Semisynthetic
Modular Biosensors
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_7, © Springer Science+Business Media LLC 2017
Chapter 7
Rational Design and Applications of Semisynthetic
Modular Biosensors: SNIFITs and LUCIDs
Helen Farrants, Julien Hiblot, Rudolf Griss, and Kai Johnsson
Abstract
Biosensors are used in many fields to measure the concentration of analytes, both in a cellular context and
in human samples for medical care. Here, we outline the design of two types of modular biosensors:
SNAP-tag-based indicators with a Fluorescent Intramolecular Tether (SNIFITs) and LUCiferase-based
Indicators of Drugs (LUCIDs). These semisynthetic biosensors quantitatively measure analyte concentrations in vitro and on cell surfaces by an intramolecular competitive mechanism. We provide an overview of
how to design and apply SNIFITs and LUCIDs.
Key words Biosensors, Protein engineering, Protein switches, Intramolecular ligands, Self-labeling
proteins, SNAP-tag, CLIP-tag, Polyproline linkers, Therapeutic drug monitoring, Cell-surface
biosensors
1 Introduction
Small molecule analytes are central to many biological systems.
The ability to quantify such analytes is important in basic research
to better understand anabolic, metabolic, and signal relay processes. It is important to quantify small molecules not only in basic
research, but also in healthcare, where it is crucial to be able to
monitor the levels of drugs and metabolites for therapeutic purposes. The use of biosensors is one method for this quantification,
and has many different applications [1, 2].
Several natural proteins bind small molecule analytes. If such a
binding protein undergoes a conformational change upon analyte
binding, it can be used as part of a biosensor system [3, 4]. For
example, it can be sandwiched in between two fluorescent proteins
that are Förster resonance energy transfer (FRET)-partners [5, 6]
or between two bioluminescent resonance energy transfer (BRET)partners [7]. In both cases, the change in distance and orientation
between the two resonance energy transfer (RET) partners is translated into changes in the optical properties of the system, which
1.1 SNIFITS
and LUCIDs:
Semisynthetic
Modular Biosensors