201
engineer genetically encoded, active site-directed competitive inhibitors
that can be directly fused to the protease transducer. Depending
on their size, individual cleavage products can either be appended
to the N- or C-terminus. Considering the N-terminal cleavage
product of TVMV comprises five of six amino acids of the core
substrate motif, this means the product-based competitive inhibitor
needs to be appended to the C-terminus. In many cases, appending a
product-based inhibitor is however insufficient to ensure a high
enough level of competitive autoinhibition. As a result, its binding
affinity needs to be improved using a combination of structure-guided
protein engineering and high-throughput combinatorial screening,
for instance, by introducing additional charge and shape complementarity between the AI-domain and the protease transducer or by
restricting the orientation of the connecting linker.
In addition, a key concern is that recombinant protein expression leads to pre-mature termination of translation and therefore
truncated protein products with no AI-domain. This may be further aggravated by limited nonspecific proteolysis of exposed
and/or flexible linkers during protein expression and/or subsequent cell lysis. This is particularly problematic for modularly
organized protease switches that rely on C-terminal AI-domains
as this limits the extent to which a synthetic protein switch is
autoinhibited in the basal state. For instance, if 10% of a purified
protein is truncated with no AI-domain, its maximum activation
is limited to tenfold. For C-terminally positioned AI-domains, it
is therefore paramount to purify full-length protein through an
affinity tag that is located C-terminal of the AI-domain. This also
requires bridging the P1-P1′ junction through a dipeptide motif
that binds, but at the same time cannot be cleaved. This is however not trivial for proteases. Bridging the P1-P1′ junction
through an uncleavable dipeptide motif also allows to place the
AI-domain in the N-terminal position and engineer additional
affinity features beyond the core cleavage product to enhance
binding. An N-terminal AI-domain also facilitates the detection
of weakly enhancing mutations, especially if their contribution
toward affinity is incremental and thus difficult to resolve if the
induction is limited by prematurely truncated protein expression
products in cell lysates. Consequently, it is recommended to first
engineer dipeptide motifs that can bridge the P1-P1′ junction
before improving the affinity of AI-domain for its cognate protease
receptor.
2 Materials
1. 100 mg/mL carbenicillin (1000× stock).
2. 50 mg/mL kanamycin (1000× stock).
3. 34 mg/mL chloramphenicol (1000× stock).
2.1 General
Reagents
Engineering Synthetic Protease Switches
engineer genetically encoded, active site-directed competitive inhibitors
that can be directly fused to the protease transducer. Depending
on their size, individual cleavage products can either be appended
to the N- or C-terminus. Considering the N-terminal cleavage
product of TVMV comprises five of six amino acids of the core
substrate motif, this means the product-based competitive inhibitor
needs to be appended to the C-terminus. In many cases, appending a
product-based inhibitor is however insufficient to ensure a high
enough level of competitive autoinhibition. As a result, its binding
affinity needs to be improved using a combination of structure-guided
protein engineering and high-throughput combinatorial screening,
for instance, by introducing additional charge and shape complementarity between the AI-domain and the protease transducer or by
restricting the orientation of the connecting linker.
In addition, a key concern is that recombinant protein expression leads to pre-mature termination of translation and therefore
truncated protein products with no AI-domain. This may be further aggravated by limited nonspecific proteolysis of exposed
and/or flexible linkers during protein expression and/or subsequent cell lysis. This is particularly problematic for modularly
organized protease switches that rely on C-terminal AI-domains
as this limits the extent to which a synthetic protein switch is
autoinhibited in the basal state. For instance, if 10% of a purified
protein is truncated with no AI-domain, its maximum activation
is limited to tenfold. For C-terminally positioned AI-domains, it
is therefore paramount to purify full-length protein through an
affinity tag that is located C-terminal of the AI-domain. This also
requires bridging the P1-P1′ junction through a dipeptide motif
that binds, but at the same time cannot be cleaved. This is however not trivial for proteases. Bridging the P1-P1′ junction
through an uncleavable dipeptide motif also allows to place the
AI-domain in the N-terminal position and engineer additional
affinity features beyond the core cleavage product to enhance
binding. An N-terminal AI-domain also facilitates the detection
of weakly enhancing mutations, especially if their contribution
toward affinity is incremental and thus difficult to resolve if the
induction is limited by prematurely truncated protein expression
products in cell lysates. Consequently, it is recommended to first
engineer dipeptide motifs that can bridge the P1-P1′ junction
before improving the affinity of AI-domain for its cognate protease
receptor.
2 Materials
1. 100 mg/mL carbenicillin (1000× stock).
2. 50 mg/mL kanamycin (1000× stock).
3. 34 mg/mL chloramphenicol (1000× stock).
2.1 General
Reagents
Engineering Synthetic Protease Switches