15
their signal either through enzymes or FPs, the preferred readouts
are based on spectroscopic assays monitoring changes in fluorescence, luminescence, or absorbance.
In addition, synthetic protein switches are usually composed of
multiple protein domains. This constitutes a frequently underestimated factor that imposes constraints on the recombinant expression of a particular class of protein switches as well as their operating
environment that both have to be accounted for in the design process. For instance, if a particular protein switch is designed to function intracellularly, its performance can be limited by cell intrinsic
factors: e.g., incomplete translation or proteolytic cleavage of flexible linker regions can limit the expression of a full-length synthetic
protein switch and ultimately the maximum induction ratio. This
constitutes less of a concern if a synthetic protein switch is developed for in vitro applications where full-length proteins can be
purified through N- and/or C-terminal purification tags.
Spectroscopic assays in combination with multiwell plate readers
constitute one of the most ubiquitous assay formats used to monitor and measure binding or catalytic functions of several thousands
of mutants by means of comparatively inexpensive and widespread
laboratory equipment. Notably, spectroscopic assays that monitor
changes in fluorescence or absorbance in multiwell plate assays formats allow for the time resolved measurement of protein function
and the possibility to duplicate samples within a single plate. The
latter greatly facilitates quantitative comparisons between synthetic
protein switches in the presence and absence of a desired target
analyte (e.g., binding ligand, cofactor, or any other target analyte
that modulates the activity of the protein switch). Colony screens
are conceptually similar to multiwell plate assays considering
microbial colonies on an agar plate comprise thousands of mutants
that can be screened on average in a cost-efficient manner. The
only added complication is that assay readouts need to be spatially
confined to individual colonies, for instance, through a FP or precipitating products of an enzyme-catalyzed reaction.
To assess the function of synthetic protein switches in high
throughput in either multiwell or colony-based screening formats,
experimental screening procedures need to be as simple as possible, ideally requiring only the sequential addition of reagents with
no successive washing steps that can introduce comparatively large
variabilities. Frequently, the target analyte or substrate cannot be
coexpressed nor readily diffuses across the cell membrane, but
needs to be added exogenously while a protein needs to be secreted
or released into the lysate. The former imposes limitations on the
functional folding of a protein, for instance, if a particular scaffold
or enzyme naturally folds in the reducing environment of the cytoplasm, it may not efficiently export and fold in the periplasm of
Escherichia coli.
4.3 Multi-cell
Screening in
Colony- and MultiwellFormat
Engineering Synthetic Protein Switches
their signal either through enzymes or FPs, the preferred readouts
are based on spectroscopic assays monitoring changes in fluorescence, luminescence, or absorbance.
In addition, synthetic protein switches are usually composed of
multiple protein domains. This constitutes a frequently underestimated factor that imposes constraints on the recombinant expression of a particular class of protein switches as well as their operating
environment that both have to be accounted for in the design process. For instance, if a particular protein switch is designed to function intracellularly, its performance can be limited by cell intrinsic
factors: e.g., incomplete translation or proteolytic cleavage of flexible linker regions can limit the expression of a full-length synthetic
protein switch and ultimately the maximum induction ratio. This
constitutes less of a concern if a synthetic protein switch is developed for in vitro applications where full-length proteins can be
purified through N- and/or C-terminal purification tags.
Spectroscopic assays in combination with multiwell plate readers
constitute one of the most ubiquitous assay formats used to monitor and measure binding or catalytic functions of several thousands
of mutants by means of comparatively inexpensive and widespread
laboratory equipment. Notably, spectroscopic assays that monitor
changes in fluorescence or absorbance in multiwell plate assays formats allow for the time resolved measurement of protein function
and the possibility to duplicate samples within a single plate. The
latter greatly facilitates quantitative comparisons between synthetic
protein switches in the presence and absence of a desired target
analyte (e.g., binding ligand, cofactor, or any other target analyte
that modulates the activity of the protein switch). Colony screens
are conceptually similar to multiwell plate assays considering
microbial colonies on an agar plate comprise thousands of mutants
that can be screened on average in a cost-efficient manner. The
only added complication is that assay readouts need to be spatially
confined to individual colonies, for instance, through a FP or precipitating products of an enzyme-catalyzed reaction.
To assess the function of synthetic protein switches in high
throughput in either multiwell or colony-based screening formats,
experimental screening procedures need to be as simple as possible, ideally requiring only the sequential addition of reagents with
no successive washing steps that can introduce comparatively large
variabilities. Frequently, the target analyte or substrate cannot be
coexpressed nor readily diffuses across the cell membrane, but
needs to be added exogenously while a protein needs to be secreted
or released into the lysate. The former imposes limitations on the
functional folding of a protein, for instance, if a particular scaffold
or enzyme naturally folds in the reducing environment of the cytoplasm, it may not efficiently export and fold in the periplasm of
Escherichia coli.
4.3 Multi-cell
Screening in
Colony- and MultiwellFormat
Engineering Synthetic Protein Switches