174
We typically measure enzyme activity by subtracting the
OD492 value of read 3 from read 5 to determine substrate
turnover/time (see Note 8).
1. The protease exclusion assay is carried out as above with the
important exception that the macromolecular binder is added
to the sensor and allowed to bind before the protease is added.
This prevents premature cleavage of the sensor by protease
before the macromolecule has an opportunity to bind and protect the sensor.
2. The typical reaction is set up as follows (see Note 9):
3. Add a suitable volume of PBS that will eventually make up a total
reaction volume of 25 μL (see step 4 in Subheading 3.3.1).
4. Add 2 μL of diluted sensor to a final concentration of approximately 5 nM and mix with 2–5 μL of a macromolecular binder.
This should be in excess of the sensor concentration to fully
block all sensor molecules. The exact ratio will depend on the
K d and should be confirmed empirically. Ideally, these components should be part of a master mix.
5. Add 2 μL of the macromolecular antagonist. The concentration of the antagonist should be at least as much as the macromolecule to allow complete dissociation.
6. After adding the macromolecular binder, incubate for 5–10 min
to allow an equilibrium to be reached (see Notes 10 and 11).
7. Add 2 μL of protease. The optimal concentration needs to be
determined empirically, e.g., for enterokinase we found
~0.15 nM to be suitable.
8. Initiate the enzyme reaction by adding 5–7 μL 1 mM nitrocefin substrate stock solution to a final concentration of 250 μM
(see Notes 6 and 7).
9. Analyze the reaction by plotting the rate of substrate turnover
against the concentration of the antagonist or blocking
macromolecule.
4 Notes
1. In some cases, the affinity of BLIP D49A for Tem1 may be
modulated for improved performance. So reverting BLIP to WT
or introducing additional mutations like W150A may be helpful.
Wang et al. [19] and Reichmann et al. [20] provide a spectrum
of mutations that could be tested for this purpose.
5. This concept can be extended to enzyme-inhibitor pairs other
than Tem1-BLIP. When constructing alternative enzymelinker- inhibitor ORFs, we would recommend placing purifica3.3.2 Protease
Exclusion Assay
Hui Chin Goh et al.
We typically measure enzyme activity by subtracting the
OD492 value of read 3 from read 5 to determine substrate
turnover/time (see Note 8).
1. The protease exclusion assay is carried out as above with the
important exception that the macromolecular binder is added
to the sensor and allowed to bind before the protease is added.
This prevents premature cleavage of the sensor by protease
before the macromolecule has an opportunity to bind and protect the sensor.
2. The typical reaction is set up as follows (see Note 9):
3. Add a suitable volume of PBS that will eventually make up a total
reaction volume of 25 μL (see step 4 in Subheading 3.3.1).
4. Add 2 μL of diluted sensor to a final concentration of approximately 5 nM and mix with 2–5 μL of a macromolecular binder.
This should be in excess of the sensor concentration to fully
block all sensor molecules. The exact ratio will depend on the
K d and should be confirmed empirically. Ideally, these components should be part of a master mix.
5. Add 2 μL of the macromolecular antagonist. The concentration of the antagonist should be at least as much as the macromolecule to allow complete dissociation.
6. After adding the macromolecular binder, incubate for 5–10 min
to allow an equilibrium to be reached (see Notes 10 and 11).
7. Add 2 μL of protease. The optimal concentration needs to be
determined empirically, e.g., for enterokinase we found
~0.15 nM to be suitable.
8. Initiate the enzyme reaction by adding 5–7 μL 1 mM nitrocefin substrate stock solution to a final concentration of 250 μM
(see Notes 6 and 7).
9. Analyze the reaction by plotting the rate of substrate turnover
against the concentration of the antagonist or blocking
macromolecule.
4 Notes
1. In some cases, the affinity of BLIP D49A for Tem1 may be
modulated for improved performance. So reverting BLIP to WT
or introducing additional mutations like W150A may be helpful.
Wang et al. [19] and Reichmann et al. [20] provide a spectrum
of mutations that could be tested for this purpose.
5. This concept can be extended to enzyme-inhibitor pairs other
than Tem1-BLIP. When constructing alternative enzymelinker- inhibitor ORFs, we would recommend placing purifica3.3.2 Protease
Exclusion Assay
Hui Chin Goh et al.