198
Proteases are also increasingly applied in biotechnology as
therapeutic and diagnostic reagents, or in basic research as molecular reporters and tools in the production of recombinant proteins.
In addition, proteases are finding increasing application in synthetic
biology as components of synthetic signaling circuits that equip
cells with new tailor-engineered functions [2, 3]. The key advantage here is the fact that, by virtue of cleaving a peptide bond, they
can be readily interfaced with any protein-associated biological
function. Notably, in comparison to alternative posttranslational
protein modifications such as protein phosphorylation, proteolysis
constitutes a drastic conformational change as one polypeptide is
split into two with the two resultant polypeptides transitioning
from an intra- to an intermolecular state. This transition is associated with a significant change in free energy that can be readily
exploited to engineer protease-inducible actuators: e.g. to activate
protein function by cleaving off an autoinhibitory (AI) domain, or
to inhibit protein function by cleaving functionally important residues. Consequently, a capacity to engineer synthetic protease
receptors in a systematic fashion constitutes a powerful tool to
engineer biological signaling transduction systems. The following
protocol chapter gives a detailed guide how to engineer and characterize synthetic protease switches with custom response functions focusing on four elementary types of signaling sensing and
transducing mechanisms: This includes autoinhibited protease
transducers, allosteric protease receptors, integrated sensing and
amplification circuits, as well as proximity-dependent protease
sensors (Fig. 1).
Elementary protease transducers, allosteric protease receptors, and
proximity-dependent protease sensors are engineered by recombining naturally occurring proteases with binding receptors and
AI-domains that bind and block the active site of the protease and
thus shut down its activity in the basal state. Individual components can either be derived from natural sources or be artificially
engineered. Given the modular organization of components, the
construction of synthetic protease switches typically occurs in a
step-wise fashion. This means, protease transducers, their cognate
AI-domains, and binding receptors can be first engineered and
characterized individually before they are assembled into functional switches, while empirically optimizing the structure and
length of the linkers connecting individual domains.
In our initial proof-of-concept studies, we chose established
proteases based on members of the Nuclear Inclusion a (NIa) family. The most prominent members are Tobacco Etch Virus (TEV)
and Tobacco Vein Mottling Virus (TVMV) protease that have
found widespread applications as tools in molecular and cell
biological research [4]. In addition, we chose the NS3 protease of
Hepatitis C Virus (HCV) which has been extensively studied as a
1.2 Choice
of Proteases
as Elementary Signal
Transducers
Viktor Stein and Kirill Alexandrov
Proteases are also increasingly applied in biotechnology as
therapeutic and diagnostic reagents, or in basic research as molecular reporters and tools in the production of recombinant proteins.
In addition, proteases are finding increasing application in synthetic
biology as components of synthetic signaling circuits that equip
cells with new tailor-engineered functions [2, 3]. The key advantage here is the fact that, by virtue of cleaving a peptide bond, they
can be readily interfaced with any protein-associated biological
function. Notably, in comparison to alternative posttranslational
protein modifications such as protein phosphorylation, proteolysis
constitutes a drastic conformational change as one polypeptide is
split into two with the two resultant polypeptides transitioning
from an intra- to an intermolecular state. This transition is associated with a significant change in free energy that can be readily
exploited to engineer protease-inducible actuators: e.g. to activate
protein function by cleaving off an autoinhibitory (AI) domain, or
to inhibit protein function by cleaving functionally important residues. Consequently, a capacity to engineer synthetic protease
receptors in a systematic fashion constitutes a powerful tool to
engineer biological signaling transduction systems. The following
protocol chapter gives a detailed guide how to engineer and characterize synthetic protease switches with custom response functions focusing on four elementary types of signaling sensing and
transducing mechanisms: This includes autoinhibited protease
transducers, allosteric protease receptors, integrated sensing and
amplification circuits, as well as proximity-dependent protease
sensors (Fig. 1).
Elementary protease transducers, allosteric protease receptors, and
proximity-dependent protease sensors are engineered by recombining naturally occurring proteases with binding receptors and
AI-domains that bind and block the active site of the protease and
thus shut down its activity in the basal state. Individual components can either be derived from natural sources or be artificially
engineered. Given the modular organization of components, the
construction of synthetic protease switches typically occurs in a
step-wise fashion. This means, protease transducers, their cognate
AI-domains, and binding receptors can be first engineered and
characterized individually before they are assembled into functional switches, while empirically optimizing the structure and
length of the linkers connecting individual domains.
In our initial proof-of-concept studies, we chose established
proteases based on members of the Nuclear Inclusion a (NIa) family. The most prominent members are Tobacco Etch Virus (TEV)
and Tobacco Vein Mottling Virus (TVMV) protease that have
found widespread applications as tools in molecular and cell
biological research [4]. In addition, we chose the NS3 protease of
Hepatitis C Virus (HCV) which has been extensively studied as a
1.2 Choice
of Proteases
as Elementary Signal
Transducers
Viktor Stein and Kirill Alexandrov