169
macromolecule like a protein domain to the peptide in the linker
sterically hinders binding of the inhibitor to the read-out enzyme
(Fig. 1). Thus, the presence of a peptide-interacting macromolecule stabilizes the open state of the read-out enzyme and increases
its activity. It is important that the flexible linker is sufficiently long
to allow the inhibitor to bind to the read-out enzyme, but not long
enough to allow a macromolecule to bind to the peptide without
sterically hindering the interaction between the read-out enzyme
and the inhibitor. In this case, the exact linker length requires
empirical optimization. Sensitivity can also be enhanced in the case
of bivalently binding macromolecules such as antibodies [13]. For
instance, this can be achieved by placing a second protein-interacting
peptide sequence in the sensor that can bind one arm of the antibody molecule and thus tether the other arm in close proximity of
the peptide in the linker. In this way, its local concentration is
increased and thus leads to increased sensitivity.
We have also developed another, more modular peptide- protein
interaction detection methodology [14]. This involves a similar
architecture, but instead of relying on the macromolecule sterically
clashing with the read-out enzyme and inhibitor binding, the macromolecule binding to its cognate peptide now sterically interferes
with proteolytic cleavage at a proteolytic cleavage site in close proximity to the peptide of interest. Thus, macromolecule binding prevents sensor activation by the protease. This system is particularly
useful for drug screening as peptide-protein antagonist drugs will
restore protease access and lead to signal generation [14].
Below is a detailed description of the construction and use of
these sensors.
2 Materials
1. Expression constructs can either be synthesized commercially
(Genscript) or custom cloned.
2. A plasmid coding for Tem1-BLIP D49A (see Note 1) inserted
into the NdeI and XhoI sites of a pET28a vector with a
C-terminal His6-tag (see Note 2). Here, BLIP stands for
β-lactamase inhibitor protein while D49A refers to a mutation
in BLIP that reduces affinity for Tem1 [15]. The coding DNA
sequence for a fusion of thermostable Tem1 β-lactamase
enzyme [16] and BLIP which is used as a template for inserting the desired peptide sequence between Tem1 and BLIP,
and if necessary at the N- or C-termini as well, is given (see
Note 3). Also, it is advisable to avoid Amp
R
cassette containing
vectors (see Note 4).
3. Standard reagents for high-fidelity PCR amplification: 10×
reaction buffer, 10 mM each dNTP solution, and a thermo2.1 Cloning
of Expression
Constructs
Sensing by Allosteric Derepression
macromolecule like a protein domain to the peptide in the linker
sterically hinders binding of the inhibitor to the read-out enzyme
(Fig. 1). Thus, the presence of a peptide-interacting macromolecule stabilizes the open state of the read-out enzyme and increases
its activity. It is important that the flexible linker is sufficiently long
to allow the inhibitor to bind to the read-out enzyme, but not long
enough to allow a macromolecule to bind to the peptide without
sterically hindering the interaction between the read-out enzyme
and the inhibitor. In this case, the exact linker length requires
empirical optimization. Sensitivity can also be enhanced in the case
of bivalently binding macromolecules such as antibodies [13]. For
instance, this can be achieved by placing a second protein-interacting
peptide sequence in the sensor that can bind one arm of the antibody molecule and thus tether the other arm in close proximity of
the peptide in the linker. In this way, its local concentration is
increased and thus leads to increased sensitivity.
We have also developed another, more modular peptide- protein
interaction detection methodology [14]. This involves a similar
architecture, but instead of relying on the macromolecule sterically
clashing with the read-out enzyme and inhibitor binding, the macromolecule binding to its cognate peptide now sterically interferes
with proteolytic cleavage at a proteolytic cleavage site in close proximity to the peptide of interest. Thus, macromolecule binding prevents sensor activation by the protease. This system is particularly
useful for drug screening as peptide-protein antagonist drugs will
restore protease access and lead to signal generation [14].
Below is a detailed description of the construction and use of
these sensors.
2 Materials
1. Expression constructs can either be synthesized commercially
(Genscript) or custom cloned.
2. A plasmid coding for Tem1-BLIP D49A (see Note 1) inserted
into the NdeI and XhoI sites of a pET28a vector with a
C-terminal His6-tag (see Note 2). Here, BLIP stands for
β-lactamase inhibitor protein while D49A refers to a mutation
in BLIP that reduces affinity for Tem1 [15]. The coding DNA
sequence for a fusion of thermostable Tem1 β-lactamase
enzyme [16] and BLIP which is used as a template for inserting the desired peptide sequence between Tem1 and BLIP,
and if necessary at the N- or C-termini as well, is given (see
Note 3). Also, it is advisable to avoid Amp
R
cassette containing
vectors (see Note 4).
3. Standard reagents for high-fidelity PCR amplification: 10×
reaction buffer, 10 mM each dNTP solution, and a thermo2.1 Cloning
of Expression
Constructs
Sensing by Allosteric Derepression