13
4 Testing Synthetic Protein Switches
Historically, biotechnological innovation has extensively relied
on experimental trial-and-error to adopt and reengineer existing
biological functions toward specific applications. This particularly
applies to the rational engineering of protein-associated functions
which has been hampered by an insufficient understanding how
the sequence of a protein relates to its function. Consequently, an
increasing number of studies are breaking down the construction
of synthetic protein switches into manageable substeps. This
includes limited empirical optimization to engineer or optimize
key functional properties such as the binding specificity of receptors and AI-domains, as well as their subsequent assembly into
functional protein switches with tailored response functions. The
latter is generally supported by medium- and high-throughput
screening assays based on multi- and single-cell assay
technologies.
The construction of modularly organized receptors and actuators,
where allosteric transitions are primarily mediated by flexible linker
regions, has raised the possibility of constructing synthetic protein
switches from individual subcomponents based on structurally
well-defined binding domains that either recognize the target
ligand or modulate the output of the actuator. For instance, GFP
and its engineered derivatives have a propensity of dimerizing with
μM affinity which has been shown to enhance the sensitivity and
dynamic range of FRET-based fluorescent sensors [115, 116].
Similarly, a number of enzymes feature naturally occurring, genetically encoded inhibitors that can be exploited for the construction
of synthetic protein switches based on the autoinhibited β-lactamase
module [56, 117, 118]. In the absence of structural information
or the presence of naturally occurring receptor and AI-domains,
highly specific protein-based binders that either recognize the target molecule or associate with the actuator to modulate its function can either be constructed de novo or sourced from natural
sources and optimized using a variety of display technologies such
as phage [119], yeast [120, 121], and various in vitro display technologies that either feature RNA [122–124] or DNA [125–129]
as the coding nucleic acid.
Collectively, these systems display a protein either on the surface of either phage or yeast or in vitro directly on its coding nucleic
acid maintaining a physical association between genotype (i.e., its
coding nucleic acid) and phenotype (i.e., the protein binder that
mediates its binding function). Depending on the type of display
system, the target ligand can be immobilized on a solid surface
retaining and enriching those phage or nucleic acids that code for
a functional binder. Alternatively, the target ligand can be labeled
with a fluorescent reporter molecule labeling those cells or μ-beads
4.1 Engineering
Subcomponents Using
Display Technologies
Engineering Synthetic Protein Switches
4 Testing Synthetic Protein Switches
Historically, biotechnological innovation has extensively relied
on experimental trial-and-error to adopt and reengineer existing
biological functions toward specific applications. This particularly
applies to the rational engineering of protein-associated functions
which has been hampered by an insufficient understanding how
the sequence of a protein relates to its function. Consequently, an
increasing number of studies are breaking down the construction
of synthetic protein switches into manageable substeps. This
includes limited empirical optimization to engineer or optimize
key functional properties such as the binding specificity of receptors and AI-domains, as well as their subsequent assembly into
functional protein switches with tailored response functions. The
latter is generally supported by medium- and high-throughput
screening assays based on multi- and single-cell assay
technologies.
The construction of modularly organized receptors and actuators,
where allosteric transitions are primarily mediated by flexible linker
regions, has raised the possibility of constructing synthetic protein
switches from individual subcomponents based on structurally
well-defined binding domains that either recognize the target
ligand or modulate the output of the actuator. For instance, GFP
and its engineered derivatives have a propensity of dimerizing with
μM affinity which has been shown to enhance the sensitivity and
dynamic range of FRET-based fluorescent sensors [115, 116].
Similarly, a number of enzymes feature naturally occurring, genetically encoded inhibitors that can be exploited for the construction
of synthetic protein switches based on the autoinhibited β-lactamase
module [56, 117, 118]. In the absence of structural information
or the presence of naturally occurring receptor and AI-domains,
highly specific protein-based binders that either recognize the target molecule or associate with the actuator to modulate its function can either be constructed de novo or sourced from natural
sources and optimized using a variety of display technologies such
as phage [119], yeast [120, 121], and various in vitro display technologies that either feature RNA [122–124] or DNA [125–129]
as the coding nucleic acid.
Collectively, these systems display a protein either on the surface of either phage or yeast or in vitro directly on its coding nucleic
acid maintaining a physical association between genotype (i.e., its
coding nucleic acid) and phenotype (i.e., the protein binder that
mediates its binding function). Depending on the type of display
system, the target ligand can be immobilized on a solid surface
retaining and enriching those phage or nucleic acids that code for
a functional binder. Alternatively, the target ligand can be labeled
with a fluorescent reporter molecule labeling those cells or μ-beads
4.1 Engineering
Subcomponents Using
Display Technologies
Engineering Synthetic Protein Switches