203
7. 1000× trace metal solution: 50 mM FeCl 3 , 20 mM CaCl 2, 10 mM
MnCl 2 , 10 mM ZnSO 4 , 2 mM CoCl 2 , 2 mM CuCl 2 , 2 mM
NiCl 2 , 2 mM Na 2 MoO 4 , 2 mM Na 2 SeO 3 , 2 mM H 3 BO 3 .
8. P-5052 autoinduction medium: 1× 5052 medium, 1× NPS,
1× trace metal and 1 mM MgSO 4 diluted in ddH 2 O.
9. 200 μg/mL tetracycline dissolved in ethanol (1000× stock).
10. P-5052 autolysis medium: 1× 5052 medium, 1× NPS and
1 mM MgSO 4 diluted in MQ supplemented with 200 ng/mL
tetracycline.
1. Terrific broth (TB): 1.2% (w/v) tryptone, 2.4% (wt/vol) yeast
extract, 0.04% glycerol, 0.17 M KH 2 PO 4 , and 0.72 M K 2 HPO 4 .
2. TB-based autoinduction medium: TB supplemented with 0.2%
lactose, 0.05% glucose, and 2 mM MgCl 2 .
3. Phosphate buffered saline (PBS): 10 mM Na 2 HPO 4 , 2 mM
KH 2 PO 4 , 137 mM NaCl, and 2.7 mM KCl, pH 7.4.
4. Washing and binding buffer: 20 mM Na 2 HPO 4 and 20 mM
imidazole pH 8.0 supplemented with 300 mM NaCl for
TVMV
Thr
-AI protease transducers, 500 mM NaCl for
HCV
TVMV
- AI protease transducers, and 1 M NaCl for TVMVFN3- PDZ-AI allosteric receptors.
5. Elution buffer: 20 mM Na 2 HPO 4 and 500 mM imidazole
pH 8.0 supplemented with 300 mM NaCl for TVMV
Thr
-AI
protease transducers, 500 mM NaCl for HCV
TVMV
-AI, and
1 M for TVMV-FN3-PDZ-AI allosteric receptors.
6. Protein storage buffer: 50 mM Tris–HCl and 10% (v/v)
glycerol, pH 8.0 supplemented with 1 mM EDTA and 2 mM
DTT for TVMV
Thr
-AI protease transducers, 1 M NaCl, 1 mM
EDTA, and 2 mM DTT for TVMV-FN3-PDZ-AI allosteric
receptors, and 500 mM NaCl and 2 mM β-mercaptoethanol
for HCV
TVMV
-AI protease transducers.
7. 0.25-μm nitrocellulose filters.
8. Ni-NTA columns (e.g., 5 mL HisTrap FF Crude from GE
Healthcare).
9. Centrifugal filters with 10-kDa cutoff.
10. PD-10 desalting columns.
1. Protease Assay Buffer: 50 mM Tris–HCl, 100 mM NaCl,
50 μg/mL BSA and 2 mM DTT, pH 8.0 (see Note 1).
2. Summary of protease reagents and their working concentrations is given in Table 1.
3. Summary of protease peptide substrate, ligand peptides, and
AI domains used to characterize individual protease receptors
is provided in Table 2.
2.4 Expression
and Purification
of TVMV and HCV
Protease Switches
2.5 Assay Synthetic
Protease Receptors
Engineering Synthetic Protease Switches
7. 1000× trace metal solution: 50 mM FeCl 3 , 20 mM CaCl 2, 10 mM
MnCl 2 , 10 mM ZnSO 4 , 2 mM CoCl 2 , 2 mM CuCl 2 , 2 mM
NiCl 2 , 2 mM Na 2 MoO 4 , 2 mM Na 2 SeO 3 , 2 mM H 3 BO 3 .
8. P-5052 autoinduction medium: 1× 5052 medium, 1× NPS,
1× trace metal and 1 mM MgSO 4 diluted in ddH 2 O.
9. 200 μg/mL tetracycline dissolved in ethanol (1000× stock).
10. P-5052 autolysis medium: 1× 5052 medium, 1× NPS and
1 mM MgSO 4 diluted in MQ supplemented with 200 ng/mL
tetracycline.
1. Terrific broth (TB): 1.2% (w/v) tryptone, 2.4% (wt/vol) yeast
extract, 0.04% glycerol, 0.17 M KH 2 PO 4 , and 0.72 M K 2 HPO 4 .
2. TB-based autoinduction medium: TB supplemented with 0.2%
lactose, 0.05% glucose, and 2 mM MgCl 2 .
3. Phosphate buffered saline (PBS): 10 mM Na 2 HPO 4 , 2 mM
KH 2 PO 4 , 137 mM NaCl, and 2.7 mM KCl, pH 7.4.
4. Washing and binding buffer: 20 mM Na 2 HPO 4 and 20 mM
imidazole pH 8.0 supplemented with 300 mM NaCl for
TVMV
Thr
-AI protease transducers, 500 mM NaCl for
HCV
TVMV
- AI protease transducers, and 1 M NaCl for TVMVFN3- PDZ-AI allosteric receptors.
5. Elution buffer: 20 mM Na 2 HPO 4 and 500 mM imidazole
pH 8.0 supplemented with 300 mM NaCl for TVMV
Thr
-AI
protease transducers, 500 mM NaCl for HCV
TVMV
-AI, and
1 M for TVMV-FN3-PDZ-AI allosteric receptors.
6. Protein storage buffer: 50 mM Tris–HCl and 10% (v/v)
glycerol, pH 8.0 supplemented with 1 mM EDTA and 2 mM
DTT for TVMV
Thr
-AI protease transducers, 1 M NaCl, 1 mM
EDTA, and 2 mM DTT for TVMV-FN3-PDZ-AI allosteric
receptors, and 500 mM NaCl and 2 mM β-mercaptoethanol
for HCV
TVMV
-AI protease transducers.
7. 0.25-μm nitrocellulose filters.
8. Ni-NTA columns (e.g., 5 mL HisTrap FF Crude from GE
Healthcare).
9. Centrifugal filters with 10-kDa cutoff.
10. PD-10 desalting columns.
1. Protease Assay Buffer: 50 mM Tris–HCl, 100 mM NaCl,
50 μg/mL BSA and 2 mM DTT, pH 8.0 (see Note 1).
2. Summary of protease reagents and their working concentrations is given in Table 1.
3. Summary of protease peptide substrate, ligand peptides, and
AI domains used to characterize individual protease receptors
is provided in Table 2.
2.4 Expression
and Purification
of TVMV and HCV
Protease Switches
2.5 Assay Synthetic
Protease Receptors
Engineering Synthetic Protease Switches