211
target (i.e., for TVMV
Thr
-AI include 0.005 U/μL thrombin
and for HCV
TVMV
-AI include 500 nM TVMV). Typical reaction volumes are 50–100 μL supplemented with 1–2 μL of
1 U/mL thrombin.
2. Mix 100 μL protease assay buffer with 50 μL of 2 μM of
TVMV
Thr
- AI or 2 μM of HCV
TVMV
-AI as pretreated ± thrombin or TVMV from step 1. Generally perform dilutions in protease assay buffer.
3. Alternatively, to assess the maximum induction ratio of allosteric protease receptors upon binding their cognate ligands, preincubate 50 μL of 1 μM TVMV-FN3-PDZ-AI together with
50 μL of protease assay buffer and 50 μL of 4 μM ligand B1 or
40 μM ligand B2 (see Note 9).
4. Alternatively, to assess the K D value of allosteric protease receptors for their cognate ligands, preincubate 50 μL of 40 nM of
TVMV-FN3-PDZ-AI with 50 μL protease assay buffer and
50 μL of varying concentrations of either ligand B1 or ligand
B2. To reliably determine the K D , the highest concentration of
ligand B1 or ligand B2 in the final reaction should be at least
100 times the anticipated K D (see Note 9).
5. Depending on the type of protease switch, initiate the reaction
by adding 50 μL of either TVMV or HCV protease peptide
substrate giving rise to a final concentration of 5 μM peptide
substrate in a final reaction volume of 200 μL. Generally
include a no enzyme control in 200 μL protease assay buffer to
account for excitation-dependent bleaching of the fluorophore
in the protease peptide substrate.
6. Monitor the reaction progress using a fluorescence multiwell plate
reader by monitoring the release of 7- methoxycoumarinyl-4-acetyl
from the quenched protease peptide substrate with ʎex/em of 330
and 405 nm. Synthetic protease switches based on TVMV or
HCV at a concentration of 100 nM or more generally yield a good
fluorescence signal over the course of 90 min. In contrast, synthetic protease switches assayed at a concentration of 10 nM or
less are preferably resolved over the course of 900 min. To prevent excessive bleaching of the fluorophore, no more than 45
time points should be measured under each condition. For
prolonged periods of time, it is also recommended to cover the
96-well plate with optical adhesive film to prevent evaporation
of the reaction assay.
7. When assessing maximum induction ratios, extract initial rates
from the change in fluorescence (after subtracting the background fluorescence with no enzyme). Then calculate the
induction ratio of individual TVMV- and HCV-based protease
switches in the inactive and active states by dividing the initial
rate in the presence and absence of the target analyte.
Engineering Synthetic Protease Switches
target (i.e., for TVMV
Thr
-AI include 0.005 U/μL thrombin
and for HCV
TVMV
-AI include 500 nM TVMV). Typical reaction volumes are 50–100 μL supplemented with 1–2 μL of
1 U/mL thrombin.
2. Mix 100 μL protease assay buffer with 50 μL of 2 μM of
TVMV
Thr
- AI or 2 μM of HCV
TVMV
-AI as pretreated ± thrombin or TVMV from step 1. Generally perform dilutions in protease assay buffer.
3. Alternatively, to assess the maximum induction ratio of allosteric protease receptors upon binding their cognate ligands, preincubate 50 μL of 1 μM TVMV-FN3-PDZ-AI together with
50 μL of protease assay buffer and 50 μL of 4 μM ligand B1 or
40 μM ligand B2 (see Note 9).
4. Alternatively, to assess the K D value of allosteric protease receptors for their cognate ligands, preincubate 50 μL of 40 nM of
TVMV-FN3-PDZ-AI with 50 μL protease assay buffer and
50 μL of varying concentrations of either ligand B1 or ligand
B2. To reliably determine the K D , the highest concentration of
ligand B1 or ligand B2 in the final reaction should be at least
100 times the anticipated K D (see Note 9).
5. Depending on the type of protease switch, initiate the reaction
by adding 50 μL of either TVMV or HCV protease peptide
substrate giving rise to a final concentration of 5 μM peptide
substrate in a final reaction volume of 200 μL. Generally
include a no enzyme control in 200 μL protease assay buffer to
account for excitation-dependent bleaching of the fluorophore
in the protease peptide substrate.
6. Monitor the reaction progress using a fluorescence multiwell plate
reader by monitoring the release of 7- methoxycoumarinyl-4-acetyl
from the quenched protease peptide substrate with ʎex/em of 330
and 405 nm. Synthetic protease switches based on TVMV or
HCV at a concentration of 100 nM or more generally yield a good
fluorescence signal over the course of 90 min. In contrast, synthetic protease switches assayed at a concentration of 10 nM or
less are preferably resolved over the course of 900 min. To prevent excessive bleaching of the fluorophore, no more than 45
time points should be measured under each condition. For
prolonged periods of time, it is also recommended to cover the
96-well plate with optical adhesive film to prevent evaporation
of the reaction assay.
7. When assessing maximum induction ratios, extract initial rates
from the change in fluorescence (after subtracting the background fluorescence with no enzyme). Then calculate the
induction ratio of individual TVMV- and HCV-based protease
switches in the inactive and active states by dividing the initial
rate in the presence and absence of the target analyte.
Engineering Synthetic Protease Switches