205
6. 8 mM HCV peptide substrate dissolved in DMSO and stored
at −80 °C.
7. 20 μM HCV peptide substrate diluted in protease assay buffer
and stored on ice.
8. 8 mM TVMV-DD peptide substrate dissolved in DMSO and
stored at −80 °C.
9. 800 μM TVMV-DD peptide substrate diluted in protease assay
buffer and stored on ice (see Note 2).
10. 1 U/μL thrombin dissolved in 50 mM Tris–HCl pH 8.0 and
stored at −80 °C.
11. 1 mM Ligand B1 dissolved in 50 mM Tris–HCl pH 8.0 and
stored at −80 °C.
12. 1 mM Ligand B2 dissolved in 50 mM Tris–HCl pH 8.0 and
stored at −80 °C.
13. 100 mM AI domain NH 2 -EYVRFAPGST-COOH as a synthetic
peptide dissolved in DMSO and stored at −80 °C.
14. 200 μg/mL rapamycin stock solution dissolved in ethanol and
stored at −80 °C.
15. Black 96-well plates for fluorescence measurements.
16. Optical adhesive film (to cover black 96-well plates for fluorescence spectroscopy).
1. PCR thermocycler.
2. Agarose gel electrophoresis equipment.
3. SDS-PAGE equipment.
4. Fluorescence multiwell plate reader (e.g., Biotek Synergy 4).
5. Shaking incubator to grow E. coli in liquid medium.
6. Static incubator to grow E. coli on agar plates.
7. One shot cell disruptor for E. coli cell lysis.
3 Method
Viral protease receptors are expressed as fusion proteins with
maltose- binding protein (MBP) based on pRK793 [11]. This construct has previously been developed to facilitate the expression of
the NIa protease from Tobacco Etch Virus (TEV) where MBP acts
as a molecular chaperone to enable more efficient recombinant
expression of NIa proteases in E. coli [12, 13]. To construct focused
libraries, a variety of DNA library generation and DNA assembly
techniques are available. For instance, combinatorial DNA libraries
with degenerate codons coding for different amino acids are suitable
to bridge the P1-P1′ junction or improve the affinity between the
2.6 Equipment
3.1 Cloning
Autoinhibited
Proteases Modules
Engineering Synthetic Protease Switches
6. 8 mM HCV peptide substrate dissolved in DMSO and stored
at −80 °C.
7. 20 μM HCV peptide substrate diluted in protease assay buffer
and stored on ice.
8. 8 mM TVMV-DD peptide substrate dissolved in DMSO and
stored at −80 °C.
9. 800 μM TVMV-DD peptide substrate diluted in protease assay
buffer and stored on ice (see Note 2).
10. 1 U/μL thrombin dissolved in 50 mM Tris–HCl pH 8.0 and
stored at −80 °C.
11. 1 mM Ligand B1 dissolved in 50 mM Tris–HCl pH 8.0 and
stored at −80 °C.
12. 1 mM Ligand B2 dissolved in 50 mM Tris–HCl pH 8.0 and
stored at −80 °C.
13. 100 mM AI domain NH 2 -EYVRFAPGST-COOH as a synthetic
peptide dissolved in DMSO and stored at −80 °C.
14. 200 μg/mL rapamycin stock solution dissolved in ethanol and
stored at −80 °C.
15. Black 96-well plates for fluorescence measurements.
16. Optical adhesive film (to cover black 96-well plates for fluorescence spectroscopy).
1. PCR thermocycler.
2. Agarose gel electrophoresis equipment.
3. SDS-PAGE equipment.
4. Fluorescence multiwell plate reader (e.g., Biotek Synergy 4).
5. Shaking incubator to grow E. coli in liquid medium.
6. Static incubator to grow E. coli on agar plates.
7. One shot cell disruptor for E. coli cell lysis.
3 Method
Viral protease receptors are expressed as fusion proteins with
maltose- binding protein (MBP) based on pRK793 [11]. This construct has previously been developed to facilitate the expression of
the NIa protease from Tobacco Etch Virus (TEV) where MBP acts
as a molecular chaperone to enable more efficient recombinant
expression of NIa proteases in E. coli [12, 13]. To construct focused
libraries, a variety of DNA library generation and DNA assembly
techniques are available. For instance, combinatorial DNA libraries
with degenerate codons coding for different amino acids are suitable
to bridge the P1-P1′ junction or improve the affinity between the
2.6 Equipment
3.1 Cloning
Autoinhibited
Proteases Modules
Engineering Synthetic Protease Switches