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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_3, © Springer Science+Business Media LLC 2017
Chapter 3
Construction of Protein Switches by Domain Insertion
and Directed Evolution
Lucas F. Ribeiro, Tiana D. Warren, and Marc Ostermeier
Abstract
A protein switch is a protein that changes between inactive (“off”) and active (“on”) states in response to
a biomolecule or physical signal. These switches can be created by fusing two domains in such a way that
the activity of the output domain is regulated by the input domain’s recognition of an input signal (such
as the binding of a molecule, recognition of light). Here, we describe several methods for randomly fusing
two domains to create domain insertion libraries from which protein switches can be identified by selections
and/or screens.
Key words Protein switch, Domain insertion, Circular permutation, Directed evolution
1 Introduction
Biological systems are often described as complex circuits consisting
of an interacting network of molecules. A key component of these
networks are protein switches that couple cellular functions. These
switches change their active state (output) in response to a physical
signal or binding event (input). The ability to create switches by
linking any desired input and output domains would enable the
rewiring of cellular circuitry to suit a researcher’s objective. This
rewiring has numerous potential applications such as cancer therapeutics [1], fluorescent biosensors [2–4], biomass degradation [5],
and regulators of cell signaling elements [6].
Our directed evolution strategy for constructing protein
switches involves fusion by domain insertion between two different proteins with the prerequisite input and output functions such
that the behavior of the output domain is responsive to signal
detection (e.g., a binding event) in the input domain (see Fig. 1).
The exact fusion to construct to create a switch can be difficult to
predict. Thus, a directed evolution approach is preferred in which
an insert gene is randomly inserted into an acceptor gene, to create
a library of gene fusions encoding fusion proteins. The insert gene
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_3, © Springer Science+Business Media LLC 2017
Chapter 3
Construction of Protein Switches by Domain Insertion
and Directed Evolution
Lucas F. Ribeiro, Tiana D. Warren, and Marc Ostermeier
Abstract
A protein switch is a protein that changes between inactive (“off”) and active (“on”) states in response to
a biomolecule or physical signal. These switches can be created by fusing two domains in such a way that
the activity of the output domain is regulated by the input domain’s recognition of an input signal (such
as the binding of a molecule, recognition of light). Here, we describe several methods for randomly fusing
two domains to create domain insertion libraries from which protein switches can be identified by selections
and/or screens.
Key words Protein switch, Domain insertion, Circular permutation, Directed evolution
1 Introduction
Biological systems are often described as complex circuits consisting
of an interacting network of molecules. A key component of these
networks are protein switches that couple cellular functions. These
switches change their active state (output) in response to a physical
signal or binding event (input). The ability to create switches by
linking any desired input and output domains would enable the
rewiring of cellular circuitry to suit a researcher’s objective. This
rewiring has numerous potential applications such as cancer therapeutics [1], fluorescent biosensors [2–4], biomass degradation [5],
and regulators of cell signaling elements [6].
Our directed evolution strategy for constructing protein
switches involves fusion by domain insertion between two different proteins with the prerequisite input and output functions such
that the behavior of the output domain is responsive to signal
detection (e.g., a binding event) in the input domain (see Fig. 1).
The exact fusion to construct to create a switch can be difficult to
predict. Thus, a directed evolution approach is preferred in which
an insert gene is randomly inserted into an acceptor gene, to create
a library of gene fusions encoding fusion proteins. The insert gene