A NanoLuc-Based Protease Biosensor for Highly Sensitive Detection of Intracellular Protease Activity: Applications to Apoptosis and Coronavirus Infection

Materials and reagents

Biological materials

1. HEK293T cells (ATCC, catalog number: CRL-3216)

2. BHK cells stably expressing hACE2 (parental BHK cells; JCRB Cell Bank, catalog number: JCRB9020); generated in-house using the PiggyBac transposon system; see General notes

3. SARS-CoV-2 (BSL-3); the virus stock is the same as that described in Yoshida et al. [4] and must be obtained under appropriate institutional and regulatory approval

4. Human coronavirus OC43 (BSL-2) (ATCC, catalog number: VR-1558)

5. FlipNanoLuc reporter plasmids (available via Addgene; see Table 1 for the plasmid list)

Table 1. FlipNanoLuc reporter and protease expression plasmids used in this protocol, with their Addgene accession numbers.

Note: Construct names follow the Addgene depositor records. CoVA Q5A and SA, and Casp3A D5A, are cleavage-site mutants used as negative controls. pCAG.CASP3-Myc encodes the caspase-3 protease (not a FlipNanoLuc reporter) and is used to validate the Casp3A reporter. The Q5A and SA cleavage-site mutants are available only in the SL1-containing backbone; see the note in Part 2, section B.

6. Gag-pol expression plasmid for retroviral particle production, as described in Morita et al. [5]

7. VSV-G expression plasmid for retroviral particle production, as described in Naldini et al. [6]

8. Protease expression plasmids: SARS-CoV-2 3CLpro, wild-type (pCAG.Myc-SARS2_3CL; Addgene, plasmid number: 200120) and the catalytically inactive C145A mutant [pCAG.Myc-SARS2_3CL(C145A); Addgene, plasmid number: 200121]; caspase-3, wild-type (pCAG.CASP3-Myc; Addgene, plasmid number: 200119)

9. PiggyBac transposon vector encoding hACE2, constructed in PB-CMV-MCS-EF1α-RedPuro (System Biosciences, catalog number: PB514B-2)

10. Super PiggyBac transposase expression vector (System Biosciences, catalog number: PB200A-1; currently supplied as catalog number: PB210PA-1)

11. Empty vector (pCAG-empty), constructed in-house; the pCAG backbone without an insert was used as the mock control in Tables 2 and 3

Table 2. Plasmid combinations for the 3CLpro (SARS-CoV-2) overexpression assay.

In every condition, 0.5 μg of reporter plasmid and 0.5 μg of co-transfected plasmid are used per well (1:1, w/w; 1 μg of total DNA).

Note: The Q5A and SA cleavage-site mutants exist only in the SL1-containing backbone. For cleavage-site specificity experiments, therefore, use the SL1-containing WT reporter (number: 200126) as the reference and compare it with the SL1-containing Q5A and SA reporters (numbers: 200127 and 200128), so that the constructs differ only in the protease recognition sequence. Do not compare the mutant reporters directly with the reporter used in the other conditions (number: 200125), which lacks SL1, because the two constructs differ in their 5′UTR and therefore in the level of reporter expression.

Table 3. Plasmid combinations for the caspase-3 (apoptosis) overexpression assay.

In every condition, 0.5 μg of reporter plasmid and 0.5 μg of co-transfected plasmid are used per well (1:1, w/w; 1 μg of total DNA).

Cell culture reagents

1. Dulbecco's modified Eagle medium (DMEM), high glucose (Nacalai Tesque, catalog number: 08458-16)

2. Minimum essential medium (MEM) (Sigma-Aldrich, catalog number: M4655)

3. Fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, catalog number: 10270-106); heat-inactivate before use (see Recipes)

Note: This catalog number is currently listed as unavailable by the supplier; an equivalent qualified FBS may be used.

4. Benzylpenicillin potassium (Fujifilm Wako Pure Chemical Corporation, catalog number: 021-07732)

5. Streptomycin sulfate (Tokyo Chemical Industry, catalog number: S0585)

6. Puromycin dihydrochloride (InvivoGen, catalog number: ant-pr-1)

7. Opti-MEM I reduced serum medium (Thermo Fisher Scientific, catalog number: 31985070)

8. Phosphate-buffered saline (PBS), without calcium and magnesium (Nacalai Tesque, catalog number: 14249-24)

9. Trypsin-EDTA solution (0.25%) (Nacalai Tesque, catalog number: 32777-15)

Transfection reagents

1. Polyethylenimine (PEI MAX, MW 40,000; 1 mg/mL stock in water) (Polysciences, catalog number: 24765-1)

Luminescence assay reagents

1. Nano-Glo^® luciferase assay system (Promega, catalog number: N1120)

2. Bright-Glo^TM luciferase assay system (Promega, catalog number: E2620)

Lysis buffer components

1. NaCl (Nacalai Tesque, catalog number: 31320-05)

2. Tris (Tris[hydroxymethyl]aminomethane) (Nacalai Tesque, catalog number: 35406-91)

3. Triton X-100 (Nacalai Tesque, catalog number: 35501-15)

4. cOmplete^TM Mini protease inhibitor cocktail tablets (Roche, catalog number: 11836153001)

5. Hydrochloric acid (HCl), for pH adjustment

6. Distilled water (used for the preparation of buffers and stock solutions)

Apoptosis assay

1. Staurosporine (AdipoGen Life Sciences, catalog number: AG-CN2-0022; CAS 62996-74-1); prepare 200 μM stock in DMSO

2. Dimethyl sulfoxide (DMSO) (Nacalai Tesque, catalog number: 13406-55)

Solutions

1. 1× lysis buffer (see Recipes)

2. 100× penicillin G + streptomycin stock solution (see Recipes)

3. Complete culture medium (see Recipes)

Recipes

1. 1× lysis buffer

Dissolve NaCl and Tris-HCl in approximately 80% of the final volume of distilled water. Add Triton X-100 and mix until dissolved. Adjust the pH to 7.4 using HCl. Bring to the final volume. Store at 4 °C for up to one month.

Note: Add the protease inhibitor cocktail immediately before use, at one tablet per 10 mL of lysis buffer (1×).

2. 100× penicillin G + streptomycin stock solution

Dissolve 0.626 g of benzylpenicillin potassium and 1 g of streptomycin sulfate in PBS to a final volume of 100 mL. Sterilize through a 0.22 μm filter and store at 4 °C for up to 6 months; for longer storage, dispense into aliquots and keep at -20 °C. This stock is used at 1× (1:100 dilution) in the respective complete culture medium (DMEM for HEK293T cells and MEM for BHK cells stably expressing hACE2).

Note: In this protocol, Pen/Strep refers to the homemade 100× penicillin G + streptomycin stock solution described in this recipe. A commercial penicillin-streptomycin solution may be substituted at the manufacturer’s recommended working concentration.

3. Complete culture medium

Combine 500 mL of basal medium (DMEM for HEK293T cells and MEM for BHK cells stably expressing hACE2), 50 mL (final 10%) of FBS that has been heat-inactivated (56 °C, 45 min), and 5 mL (final 1×) of 100× penicillin G + streptomycin stock solution. Mix gently. Store at 4 °C for up to 2 months. For the maintenance medium of BHK cells stably expressing hACE2, additionally supplement with puromycin dihydrochloride to a final concentration of 3 μg/mL.

Laboratory supplies

1. White 384-well plates (Greiner Bio-One, catalog number: 784075)

2. 24-well cell culture plates (Violamo, catalog number: 2-8588-03)

3. 96-well cell culture plates (Violamo, catalog number: 2-8588-05)

4. 3.5 cm (35 mm) cell culture dishes (Thermo Scientific, catalog number: 130180)

5. Microcentrifuge tubes, 1.5 mL (Watson, catalog number: 131-815C)

6. Syringe filters, 0.22 μm (AS ONE, catalog number: 033022SO-SFCA)

Equipment

1. Varioskan LUX multimode plate reader (Thermo Fisher Scientific, catalog number: VLBL00D1) or an equivalent luminometer capable of luminescence detection in 384-well format

2. Multichannel pipette (suitable for 10 μL) (Thermo Scientific, catalog number: 4661040N)

3. Refrigerated microcentrifuge capable of 500× g at 4 °C (Thermo Scientific, catalog number: 75002559)

4. BSL-2 and BSL-3 certified biosafety cabinets (as appropriate) (HITACHI, catalog number: SCV-1303ECIIB)

5. 37 °C incubator with 5% CO₂ (PHC, catalog number: MCO-170AIC-PJ)

6. Inverted phase-contrast microscope (OLYMPUS, model: CKX53)

7. Water bath capable of 56 °C (Yamato Scientific, catalog number: BF200)

Software and datasets

1. GraphPad Prism (version 11.0.0 for macOS) (GraphPad Software, https://www.graphpad.com)

2. SkanIt Software for microplate readers (Thermo Fisher Scientific), used for data acquisition on the Varioskan LUX plate reader v. 6.0.2.3

Procedure