217
9. To measure the maximum induction ratio, the binding interaction
needs to be saturated. Given the AI-domain creates a decoy
interaction, the affinity of an allosteric receptor for its cognate
protease will be reduced.
10. For alternative protease sensors, the K M needs to be determined empirically.
11. Depending on whether the TVMV-based protease transducer
is partially autoinhibited, the signal-to-noise ratios and the
response time of the proximity-dependent protease switches
can be optimized. For instance, at high sensitivity and low concentrations of FKBP12-TVMV-AI in the sub-nM range, nonspecific cleavage and activation of the secondary amplifier is
negligible and it is preferable to employ uninhibited TVMV. In
contrast, at high concentrations of FKBP12-TVMV-AI, nonspecific activation is elevated and it is therefore recommended
to employ partially autoinhibited TVMV modules. Employing
partially autoinhibited TVMV modules generally does not
interfere, to a significant extent, with cleavage considering the
TVMV cleavage site in the secondary amplifier is presented at
a high local concentration in the ternary complex.
Acknowledgments
This work was funded by the Australian Research Council Discovery
Project Grant DP1094080 to KA and in part by National Breast
Cancer Foundation Innovator Grant. This research was also supported by Movember through Australia’s Prostate Cancer
Foundation Research Program to KA and VS.
References
1. Neil D. Rawlings GS (eds) (2013). Handbook
of proteolytic enzymes, Academic Press,
Cambridge, Massachusetts.
2. Stein V, Alexandrov K (2014) Protease-based
synthetic sensing and signal amplification.
Proc Natl Acad Sci U S A 111:15934–15939.
doi:10.1073/pnas.1405220111
3. Stein V, Alexandrov K (2015) Synthetic protein
switches: design principles and applications.
Trends Biotechnol 33:101–110. doi:10.1016/j.
tibtech.2014.11.010
4. Cesaratto F, Burrone OR, Petris G (2016)
Tobacco etch virus protease: a shortcut across
biotechnologies. J Biotechnol 231:239–249.
doi:10.1016/j.jbiotec.2016.06.012
5. Ingallinella P, Bianchi E, Ingenito R et al
(2000) Optimization of the P′-region of peptide inhibitors of hepatitis C virus NS3/4A
protease. Biochemistry 39:12898–12906.
doi:10.1021/bi001590g
6. Huang J, Makabe K, Biancalana M et al (2009)
Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated
through directed domain-interface evolution.
J Mol Biol 392:1221–1231. doi:10.1016/j.
jmb.2009.07.067
7. Huang J, Koide A, Makabe K, Koide S (2008)
Design of protein function leaps by directed
domain interface evolution. Proc Natl Acad
Sci U S A 105:6578–6583. doi:10.1073/
pnas.0801097105
8. Xu L, Li S, Ren C et al (2006) Heat-inducible
autolytic vector for high-throughput screening. Biotechniques 41:319–323. doi:10.2144/
000112219
Engineering Synthetic Protease Switches
9. To measure the maximum induction ratio, the binding interaction
needs to be saturated. Given the AI-domain creates a decoy
interaction, the affinity of an allosteric receptor for its cognate
protease will be reduced.
10. For alternative protease sensors, the K M needs to be determined empirically.
11. Depending on whether the TVMV-based protease transducer
is partially autoinhibited, the signal-to-noise ratios and the
response time of the proximity-dependent protease switches
can be optimized. For instance, at high sensitivity and low concentrations of FKBP12-TVMV-AI in the sub-nM range, nonspecific cleavage and activation of the secondary amplifier is
negligible and it is preferable to employ uninhibited TVMV. In
contrast, at high concentrations of FKBP12-TVMV-AI, nonspecific activation is elevated and it is therefore recommended
to employ partially autoinhibited TVMV modules. Employing
partially autoinhibited TVMV modules generally does not
interfere, to a significant extent, with cleavage considering the
TVMV cleavage site in the secondary amplifier is presented at
a high local concentration in the ternary complex.
Acknowledgments
This work was funded by the Australian Research Council Discovery
Project Grant DP1094080 to KA and in part by National Breast
Cancer Foundation Innovator Grant. This research was also supported by Movember through Australia’s Prostate Cancer
Foundation Research Program to KA and VS.
References
1. Neil D. Rawlings GS (eds) (2013). Handbook
of proteolytic enzymes, Academic Press,
Cambridge, Massachusetts.
2. Stein V, Alexandrov K (2014) Protease-based
synthetic sensing and signal amplification.
Proc Natl Acad Sci U S A 111:15934–15939.
doi:10.1073/pnas.1405220111
3. Stein V, Alexandrov K (2015) Synthetic protein
switches: design principles and applications.
Trends Biotechnol 33:101–110. doi:10.1016/j.
tibtech.2014.11.010
4. Cesaratto F, Burrone OR, Petris G (2016)
Tobacco etch virus protease: a shortcut across
biotechnologies. J Biotechnol 231:239–249.
doi:10.1016/j.jbiotec.2016.06.012
5. Ingallinella P, Bianchi E, Ingenito R et al
(2000) Optimization of the P′-region of peptide inhibitors of hepatitis C virus NS3/4A
protease. Biochemistry 39:12898–12906.
doi:10.1021/bi001590g
6. Huang J, Makabe K, Biancalana M et al (2009)
Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated
through directed domain-interface evolution.
J Mol Biol 392:1221–1231. doi:10.1016/j.
jmb.2009.07.067
7. Huang J, Koide A, Makabe K, Koide S (2008)
Design of protein function leaps by directed
domain interface evolution. Proc Natl Acad
Sci U S A 105:6578–6583. doi:10.1073/
pnas.0801097105
8. Xu L, Li S, Ren C et al (2006) Heat-inducible
autolytic vector for high-throughput screening. Biotechniques 41:319–323. doi:10.2144/
000112219
Engineering Synthetic Protease Switches