210
3. Grow cells over the course of 48 h at 30 °C at 200 rpm expressing the protein by means of autoinduction (see Note 7). Cells
usually reach OD 600 values of ∼14.
4. Harvest cells by centrifugation at 4500 × g and wash once with
500 mL PBS. At this stage, cells can either be stored at −20 °C
or directly processed.
5. Resuspend cells in washing and binding buffer and lyse using a
one-shot cell disruptor.
6. Centrifuge samples at 25,000 × g and pass the supernatant
through a 0.25-μm nitrocellulose filter to remove any largescale debris and genomic DNA that could clog up the affinity
purification column in the subsequent step.
7. Load the lysate onto 5 mL HisTrap FF Crude Ni-NTA columns equilibrated with washing and binding buffer.
8. After loading the cell lysate allow the His 6 -tagged protease
switch to bind to the column and then wash the column with
40 column volumes of washing and binding buffer.
9. Elute the His 6 -tagged protease switch with an imidazole gradient
from 20 to 500 mM over 40 column volumes. Throughout the
procedure maintain a flow rate of 5 mL/min. The His 6 -tagged
protease switch typically elutes around 100 mM imidazole.
10. Pool fractions that contain the His 6 -tagged protease switch,
concentrate the pooled fractions using centrifugal filters with a
10 kDa cutoff and transfer into storage buffer with a suitable
concentration of NaCl by means of gel filtration using disposable PD-10 desalting columns (see Note 8).
11. Synthetic protease switches can either be assayed directly or
stored in aliquots at −80 °C. Aliquoting samples and flashfreezing in liquid nitrogen is highly recommended to prevent
deterioration of the protein sample over successive freeze-thaw
cycles.
Once purified, individual protease receptors can be assayed and
characterized under defined reaction conditions: e.g., to assess
their maximum induction ratios, determine the dissociation constants of allosteric protease receptors for their cognate ligands,
measure the inhibition constants of AI-domain peptides, or assess
the purity of protease transducers by means of kinetic analysis. The
preferred assay reaction volume is 200 μL while individual assay
components are preferably added in increments of 50 μL based on
4× working stock solutions of individual reagents.
1. To assess the maximum induction ratio of elementary autoinhibited protease transducers, preincubate 10 μM of TVMV
Thr
- AI
or 10 μM of HCV
TVMV
-AI for 10 min in 100 μL protease assay
buffer in the presence and absence of an activating protease
3.4 Quantification
of Synthetic Protease
Switches
3.4.1 Determine
the Induction
of Autoinhibited Protease
Transducers and Receptors
Viktor Stein and Kirill Alexandrov
3. Grow cells over the course of 48 h at 30 °C at 200 rpm expressing the protein by means of autoinduction (see Note 7). Cells
usually reach OD 600 values of ∼14.
4. Harvest cells by centrifugation at 4500 × g and wash once with
500 mL PBS. At this stage, cells can either be stored at −20 °C
or directly processed.
5. Resuspend cells in washing and binding buffer and lyse using a
one-shot cell disruptor.
6. Centrifuge samples at 25,000 × g and pass the supernatant
through a 0.25-μm nitrocellulose filter to remove any largescale debris and genomic DNA that could clog up the affinity
purification column in the subsequent step.
7. Load the lysate onto 5 mL HisTrap FF Crude Ni-NTA columns equilibrated with washing and binding buffer.
8. After loading the cell lysate allow the His 6 -tagged protease
switch to bind to the column and then wash the column with
40 column volumes of washing and binding buffer.
9. Elute the His 6 -tagged protease switch with an imidazole gradient
from 20 to 500 mM over 40 column volumes. Throughout the
procedure maintain a flow rate of 5 mL/min. The His 6 -tagged
protease switch typically elutes around 100 mM imidazole.
10. Pool fractions that contain the His 6 -tagged protease switch,
concentrate the pooled fractions using centrifugal filters with a
10 kDa cutoff and transfer into storage buffer with a suitable
concentration of NaCl by means of gel filtration using disposable PD-10 desalting columns (see Note 8).
11. Synthetic protease switches can either be assayed directly or
stored in aliquots at −80 °C. Aliquoting samples and flashfreezing in liquid nitrogen is highly recommended to prevent
deterioration of the protein sample over successive freeze-thaw
cycles.
Once purified, individual protease receptors can be assayed and
characterized under defined reaction conditions: e.g., to assess
their maximum induction ratios, determine the dissociation constants of allosteric protease receptors for their cognate ligands,
measure the inhibition constants of AI-domain peptides, or assess
the purity of protease transducers by means of kinetic analysis. The
preferred assay reaction volume is 200 μL while individual assay
components are preferably added in increments of 50 μL based on
4× working stock solutions of individual reagents.
1. To assess the maximum induction ratio of elementary autoinhibited protease transducers, preincubate 10 μM of TVMV
Thr
- AI
or 10 μM of HCV
TVMV
-AI for 10 min in 100 μL protease assay
buffer in the presence and absence of an activating protease
3.4 Quantification
of Synthetic Protease
Switches
3.4.1 Determine
the Induction
of Autoinhibited Protease
Transducers and Receptors
Viktor Stein and Kirill Alexandrov