14
that display a functional binder and enriching them by means of
fluorescence activated cell sorting (FACS).
Beyond choosing a suitable display system, the second major
consideration concerns the scaffold protein to construct tailored
protein binders. Historically, the development of next- generation
biologics has yielded a diverse repertoire of recombinant binding
scaffolds as alternatives to monoclonal antibodies [130–132].
These typically comprise independently folding, single-chain protein domains and short peptide motifs with more or less defined
structural propensities. Crucially, these newly developed binding
scaffolds can be readily produced in Escherichia coli, fused to
additional protein domains and generally display superior structural, folding, and thermodynamic properties that facilitate their
purification, biophysical characterization, and integration into
modularly organized synthetic protein switches.
In one recent example, an allosteric binding receptor was
constructed by means of phage display fusing a circularly permutated PDZ domain with an engineered fibronectin (FN) scaffold
that serves as an enhancer domain [57, 59]. The two domains
are connected through a Gly-Ser rich linker, which is unstructured in the ligand unbound state, but forms a structurally welldefined sandwich complex in the ligand-bound state. Biophysical
studies have also shown that formation of the sandwich complex
is associated with a distinct movement of the receptor domain.
This was subsequently exploited to create fluorescence and protease-based switches following recombination of the affinity
clamp receptor with fluorescent proteins [58] and autoinhibited
protease modules [60].
Arguably, the most technically challenging aspect in the construction of synthetic protein switches is to recombine individual subcomponents (e.g., the binding receptor, the actuator, and
AI-domains) into fully functional protein switches with tailored
response functions. Depending on the type of switch, this requires
testing a varying number of designs over successive screening and
selection cycles while looking to optimize their input-dependent
switching behavior. Experimentally, this is the most labor-intensive step and, apart from designing a particular synthetic protein
switch (see Subheading 2), the most creative one considering for
every different actuator a tailored screening assay needs to be
devised.
As a rule of thumb, the higher the throughput, the more technically challenging it becomes to establish a suitable screening
assay. This particularly applies to synthetic protein switches that are
ideally screened in positive and negative selection modes looking
to identify those switches that display the largest differential function in the presence and absence of a desired target analyte.
Considering the majority of synthetic protein switches actuate
4.2 Assembling
Synthetic Protein
Switches
Viktor Stein
that display a functional binder and enriching them by means of
fluorescence activated cell sorting (FACS).
Beyond choosing a suitable display system, the second major
consideration concerns the scaffold protein to construct tailored
protein binders. Historically, the development of next- generation
biologics has yielded a diverse repertoire of recombinant binding
scaffolds as alternatives to monoclonal antibodies [130–132].
These typically comprise independently folding, single-chain protein domains and short peptide motifs with more or less defined
structural propensities. Crucially, these newly developed binding
scaffolds can be readily produced in Escherichia coli, fused to
additional protein domains and generally display superior structural, folding, and thermodynamic properties that facilitate their
purification, biophysical characterization, and integration into
modularly organized synthetic protein switches.
In one recent example, an allosteric binding receptor was
constructed by means of phage display fusing a circularly permutated PDZ domain with an engineered fibronectin (FN) scaffold
that serves as an enhancer domain [57, 59]. The two domains
are connected through a Gly-Ser rich linker, which is unstructured in the ligand unbound state, but forms a structurally welldefined sandwich complex in the ligand-bound state. Biophysical
studies have also shown that formation of the sandwich complex
is associated with a distinct movement of the receptor domain.
This was subsequently exploited to create fluorescence and protease-based switches following recombination of the affinity
clamp receptor with fluorescent proteins [58] and autoinhibited
protease modules [60].
Arguably, the most technically challenging aspect in the construction of synthetic protein switches is to recombine individual subcomponents (e.g., the binding receptor, the actuator, and
AI-domains) into fully functional protein switches with tailored
response functions. Depending on the type of switch, this requires
testing a varying number of designs over successive screening and
selection cycles while looking to optimize their input-dependent
switching behavior. Experimentally, this is the most labor-intensive step and, apart from designing a particular synthetic protein
switch (see Subheading 2), the most creative one considering for
every different actuator a tailored screening assay needs to be
devised.
As a rule of thumb, the higher the throughput, the more technically challenging it becomes to establish a suitable screening
assay. This particularly applies to synthetic protein switches that are
ideally screened in positive and negative selection modes looking
to identify those switches that display the largest differential function in the presence and absence of a desired target analyte.
Considering the majority of synthetic protein switches actuate
4.2 Assembling
Synthetic Protein
Switches
Viktor Stein