18
[25–27, 97, 98, 148], but also mutually exclusive binding interactions [56, 117, 118]. Saccharomyces cerevisiae also provides a powerful microorganism for devising high-throughput selection
procedures based on genetic complementation. In one recent
example, a light- regulated K
+
channel was engineered by recombining a photo- responsive LOV2-Jα domain with the small viral K
+
channel K cv [149]. To this end, synthetic K
+
channels were screened
for light- responsiveness in positive and negative selection modes
following illumination with blue light or in the dark. The selection
strategy was based on a mutant strain of Saccharomyces cerevisiae
deficient in endogenous K
+
channels. Similar genetic complementation strategies are conceivable to screen for metabolic functions
in high throughput through auxotrophic complementation of
metabolic enzymes in both Escherichia coli and Saccharomyces
cerevisiae.
Ultimately however, the key technical challenge with growthbased selection assays is to control the reaction conditions, in particular, the reaction environment, the concentration of individual
components and the selective pressure. In addition, the growth of
a particular synthetic protein switch may not exclusively depend on
its function, but a cell can both adapt genetically and biochemically
to enhance growth irrespective of a given synthetic protein switch
mutant.
5 Outlook
Synthetic protein switches are increasingly developed and applied
both in basic research and biotechnology to monitor biological
processes in an integrated and autonomous fashion. For now,
due to our limited understanding to predictively engineer proteinassociated functions, the construction of tailor-engineered protein
switches has relied, to a significant extent, on empirical optimization based on high-throughput screening procedures. Suitable
high-throughput screening and selection procedures are however
technically challenging to establish, need to be tailored toward specific enzyme readouts, and are ideally amenable to positive and
negative selection modes. This is further hampered by the vast size
of protein sequence space which generally outstrips our capacity to
screen and engineer protein-associated functions in high throughput. This particularly applies to engineering allostericity that constitutes one of the most complex and least understood protein
functions. Computational strategies have therefore been limited to
optimizing individual properties such as the thermodynamic stability or the binding specificity of an allosteric receptor, but will
undoubtedly continue gaining importance as our molecular mechanistic understanding of artificially engineered protein switches is
anticipated to improve.
Viktor Stein
[25–27, 97, 98, 148], but also mutually exclusive binding interactions [56, 117, 118]. Saccharomyces cerevisiae also provides a powerful microorganism for devising high-throughput selection
procedures based on genetic complementation. In one recent
example, a light- regulated K
+
channel was engineered by recombining a photo- responsive LOV2-Jα domain with the small viral K
+
channel K cv [149]. To this end, synthetic K
+
channels were screened
for light- responsiveness in positive and negative selection modes
following illumination with blue light or in the dark. The selection
strategy was based on a mutant strain of Saccharomyces cerevisiae
deficient in endogenous K
+
channels. Similar genetic complementation strategies are conceivable to screen for metabolic functions
in high throughput through auxotrophic complementation of
metabolic enzymes in both Escherichia coli and Saccharomyces
cerevisiae.
Ultimately however, the key technical challenge with growthbased selection assays is to control the reaction conditions, in particular, the reaction environment, the concentration of individual
components and the selective pressure. In addition, the growth of
a particular synthetic protein switch may not exclusively depend on
its function, but a cell can both adapt genetically and biochemically
to enhance growth irrespective of a given synthetic protein switch
mutant.
5 Outlook
Synthetic protein switches are increasingly developed and applied
both in basic research and biotechnology to monitor biological
processes in an integrated and autonomous fashion. For now,
due to our limited understanding to predictively engineer proteinassociated functions, the construction of tailor-engineered protein
switches has relied, to a significant extent, on empirical optimization based on high-throughput screening procedures. Suitable
high-throughput screening and selection procedures are however
technically challenging to establish, need to be tailored toward specific enzyme readouts, and are ideally amenable to positive and
negative selection modes. This is further hampered by the vast size
of protein sequence space which generally outstrips our capacity to
screen and engineer protein-associated functions in high throughput. This particularly applies to engineering allostericity that constitutes one of the most complex and least understood protein
functions. Computational strategies have therefore been limited to
optimizing individual properties such as the thermodynamic stability or the binding specificity of an allosteric receptor, but will
undoubtedly continue gaining importance as our molecular mechanistic understanding of artificially engineered protein switches is
anticipated to improve.
Viktor Stein