Protocol for production and purification of SARS-CoV-2 3CLpro

Summary 3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L−1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.1

His 8 -MBP-SUMO pro expression plasmid Plasmid pGTM_YR375_SUMO_Protease_001 ( Figure 2) was obtained by cloning the CDS for residues 347-621 of the Ubiquitin-like-specific protease 1 from Saccharomyces cerevisiae [Uniprot ID: Q02724 (ULP1_YEAST)] into the pET15_8His_MbpTEV_NESG expression vector, 7 which contains the CDS for an N-terminal octa-His tag upstream to Maltose Binding Protein Solubility Enhancing Tag (MBP tag) and TEV protease cleavage site. This plasmid has a pMB1 origin of replication and carries the ampicillin resistance gene. Plasmid pGTM_YR375_SUMO_Protease_001 is available from AddGene (AddGene ID: 190063) and the expressed protein construct will be called hereafter His 8 -MBP-SUMO pro . a. Weigh 2.38 g (or 0.238 g) of IPTG (molar mass 238.30 g mol À1 ) (Sigma Aldrich). b. Dissolve it in a final volume of 10 mL (or 1.0 mL) using MilliQ water (or 2 H 2 O). c. Sterilize using 0.22 mm syringe filters and store at À20 C as 1 mL aliquots. 3. Prepare 1 M magnesium chloride (MgCl 2 ) in H 2 O (or 2 H 2 O). a. Weigh 95.2 g (or 0.30 g) of MgCl 2 (molar mass 95.21 g mol À1 ) (Sigma Aldrich). b. Dissolve it in a final volume of 1 L (or 3.0 mL) using MilliQ water (or 2 H 2 O). c. Filter the solution using 0.45 mm syringe filters and store at room temperature (20-25 C). 4. Prepare 40 mg mL À1 deoxyribonuclease I from bovine pancreas (DNase I).

Preparation of reagent stock solutions
a. Weigh 40.0 mg of Dnase I (Sigma Aldrich). b. Dissolve it in a final volume of 1 mL using MilliQ water. c. Store at À20 C as 30 mL aliquots. 5. Prepare 1 M dithiothreitol (DTT).
a. Weigh 1.55 g of anhydrous NiSO 4 (molar mass 154.76 g mol À1 ) (Sigma Aldrich). b. Dissolve it in a final volume of 100 mL using MilliQ water. c. Filter the solution using 0.45 mm syringe filters and store at room temperature.
Note: Trace element stock for MJ9 medium can be stored for several months in frozen aliquots at À20 C.
Note: Trace Element Stock is not required when using the LB or M9 medium protocols a. Weigh 6.25 g of LB broth powder (Miller) (Sigma Aldrich) and 3.75 g of agar (Sigma Aldrich). b. Add 250 mL of MilliQ water and dissolve the powder mixture (agar will completely dissolve only after heating during autoclave sterilization -see next point). c. Sterilize in autoclave (121 C, 20 min). ll OPEN ACCESS d. Allow the sterilized LB-agar mixture to cool down to $ 50 C. e. Add 500 mL of 50 mg mL À1 ampicillin stock solution.
CRITICAL: Antibiotics must be added after cooling down to $ 50 C to prevent thermal degradation.
f. Pour the LB-agar into the Petri dishes under aseptic conditions inside the laminar flow cabinet. g. Wait until the agar solidifies and seal the plate with parafilm. h. Store the LB-agar plates at 4 C upside down, to prevent aqueous vapor condensation on the surface of the LB-Agar.
Alternatives: to obtain higher yields (up to 120 mg L À1 fermentation), other rich media (i.e. Super Broth, Research Products International) can be used in place of LB.
Preparation of medium for production of 15 N-, 13 C-or 15 N, 13

SUMO protease expression and purification
Timing: 6 days Cell transformation and DNA extraction are performed following the protocol described in the section transformation of Escherichia coli cells using plasmid pGTM_YR375_SUMO_Protease_001 instead of plasmid pGTM_CoV2_NSP5_004_SUMO. Expression of His 8 -MBP-SUMO pro , as well as cell lysis, soluble extract recovery, and a first step of immobilized metal affinity chromatography (IMAC) are performed following steps 1-3 as described in the section expression and purification of SARS-CoV-2 3CL pro , using lysis, washing, and elution buffers at pH 7.5 instead of pH 8.0. The His 8 -MBP-SUMO pro eluted from the IMAC is buffer-exchanged in a storage buffer containing 50 mM Tris-HCl, 150 mM NaCl, 10 % glycerol, 0.5 mM DTT, at adjusted pH 7.5; the expressed protease retains its proteolytic activity with no need for cleavage of the His 8 -MBP tag by TEV protease. It can be concentrated up to 1 mg mL À1 and stored in 1 mL aliquots at À80 C. It will be used at a ratios (w/w) of 1:1000 to 1:100 to cleave the His 6 -SUMO tag from His 6 -SUMO-3CL pro , to obtain native 3CL pro .

OPEN ACCESS
Note: His 8 -MBP-SUMO pro has a pI of $ 10, and its purification protocol uses a buffer at pH 7.5. However, for His 6 -SUMO-3CL pro (pI $ 5.8) and 3CL pro (pI $ 5.9), the purification protocol (described below) used a buffer at pH 8.0, which is farther from the isoelectric points of these constructs.
Note: Specific activity of His 8 -MBP-SUMO pro may vary depending on the protein batch preparation, and the optimal ratio and cleavage temperature should be empirically determined by performing cleavage assays on small scale samples. In our experience, undergoing multiple freeze/thaw cycles can reduce protease activity. Therefore, we recommend freezing the purified SUMO pro in small (e.g. 50 mL) aliquots, which can be stored for up to 3 months. Individual aliquots should be thawed immediately before needed in the 12-16 h cleavage/dialysis.

Transformation of Escherichia coli cells
Timing: 18 h 13. Transformation of XL10 Gold cells by heat shock for DNA propagation purposes 9 (Day 1).
Note: Additions of DNA and liquid medium to the cell suspension, as well as transferring and plating procedures, must be carried out working under aseptic conditions inside the laminar flow cabinet or using a Bunsen burner to prevent cell culture contamination.
a. Pre-warm 1 mL of Super Optimal broth with Catabolite repression (SOC) medium at 37 C in a 1.5 mL sterile microcentrifuge tube. b. Thaw one aliquot (50-100 mL) of XL10 Gold Escherichia coli cells on ice. c. Pipette ca. 50 ng of the pGTM_CoV2_NSP5_004_SUMO plasmid directly into the bacterial suspension. d. Mix by gently flicking the tube and incubate on ice for 30 min. e. Perform the heat shock by transferring the suspension at 42 C (use either a dry or water bath) for 60 s and then on ice for 2 min. f. Add the pre-warmed SOC medium to the suspension and incubate for 45 min at 37 C under agitation (at 225 rpm). g. Plate 100 mL of the suspension on a previously prepared LB-agar Petri dish with 100 mg mL À1 ampicillin. h. Incubate at 37 C for 16 h under agitation (at 225 rpm). 14. Plasmid propagation and DNA extraction from XL10 Gold cells (Days 2 and 3).
a. Pick a single colony from the plates prepared in step 13 and containing transformed XL10 Gold cells, inoculate it in 5 mL of SOC or LB medium with 100 mg mL À1 ampicillin and incubate at 37 C for 16 h under agitation (at 225 rpm). b. Carry out the DNA extraction by using a NucleoSpin Plasmid (NoLid) kit (Macherey-Nagel) and following manufacturer's instructions (https://www.mn-net.com/media/pdf/45/51/02/ Instruction-NucleoSpin-Plasmid.pdf, page 15). c. Plasmid DNA can be stored in aliquots at À20 C.
Alternatives: Other commercially available plasmid extraction kits may also be used. 15. Transformation of BL21(DE3) Escherichia coli cells by heat shock for protein expression purposes is carried out using the pGTM_CoV2_NSP5_004_SUMO plasmid extracted from XL10 Gold cells in the previous step, and following the protocol outlined in step 13 of this section using instead BL21(DE3) Escherichia coli cells. This will provide transformed BL21(DE3) cells in a plate that can be used for the next part of the protocol.

STEP-BY-STEP METHOD DETAILS
Expression and purification of SARS-CoV-2 3CL pro

Timing: 4 days
This section details all the experimental steps needed to express His 6 -SUMO-3CL pro from BL21(DE3) Escherichia coli cells harboring the pGTM_CoV2_NSP5_004_SUMO (or pGTM_CoV2_NSP5_-C145A_SUMO) plasmid, and to obtain 3CL pro (or C145A-3CL pro ) in a pure and native form. Prepare 0.1 L, adjust to pH 7.3, and store at 4 C *To be added just before setting up the assay ll OPEN ACCESS Note: The transferring of cell cultures, as well as the addition of reagents and the collection of cell samples for the measurement of cellular growth must be carried out working under aseptic conditions inside the laminar flow cabinet or using a Bunsen burner to prevent cell culture contamination.
a. Prepare a starter culture of transformed BL21(DE3) cells by picking and inoculating a single colony (from plates prepared at step 13 in the before you begin section, which contain transformed BL21(DE3) cells) in 50 mL of LB broth with 100 mg mL À1 ampicillin added. Incubate the resulting suspension for 16 h at 37 C under agitation (at 225 rpm). b. Transfer the starter culture to 1 L of LB broth with 100 mg mL À1 ampicillin added and incubate the suspension at 37 C under agitation (225 rpm). c. Measure the optical density at 600 nm (OD 600 ) every 30 min until it reaches ca. 0.6 units (approximately 1.5-2 h). d. Add IPTG to a final concentration of 1 mM to induce protein expression. e. Incubate for 16 h at 17 C under agitation (225 rpm).
Alternatives: To prepare isotopically-enriched samples for NMR experiments using MJ9 minimal medium optimized for isotope-enrichment, 8 at the end of point a above, transfer 25 mL of the obtained starter culture to 1 L of MJ9 minimal medium added with 50 mg mL À1 ampicillin. The resulting suspension should be incubated at 37 C under stirring (225 rpm) and treated as described from point c onwards.
Alternatives: While the best results are usually obtained using MJ9 medium, M9 minimal medium can also be used. In this case, once OD 600 reaches ca. 0.6 units (point c above), harvest the cells by centrifugation for 20 min at 7000 3 g at room temperature and resuspend the pellet in 375 mL of M9 minimal medium containing 15 N ammonium sulphate (and/or 13 C-enriched glucose) added with 50 mg mL À1 ampicillin. Incubate the resulting suspension for 20 min at 17 C under agitation (225 rpm), then induce and further incubate as described in points d and e.
Alternatives: To prepare 15 N, 13 C, 2 H-enriched samples for NMR experiments using MJ9 minimal medium optimized for isotope-enrichment, 8 fully replace step 1 a-e with the following procedure: f. Prepare a starter culture of transformed BL21(DE3) cells by picking and inoculating a single colony (from plates prepared at step 13 in the before you begin section) in 1.0 mL of LB medium added with 100 mg mL À1 ampicillin. Incubate the resulting suspension at 37 C until OD 600 reaches 0.6 units (approximately 1.5-2 h). g. Harvest the cells by centrifugation (7000 3 g) at room temperature. h. Resuspend the cell pellet in 3.0 mL of MJ9 medium A added with 50 mg mL À1 ampicillin. i. Incubate the cell suspension in a 15 mL Falcon Tube at 37 C under agitation (225 rpm) until OD 600 reaches 0.5 units (approximately 1.5-2 h).
CRITICAL: OD 600 must not exceed 0.65 units to ensure that the culture is still in the log phase.  a. Harvest the cells by centrifuging the culture for 30 min at 7000 3 g at 4 C. b. Gently resuspend the harvested cells in 20 mL lysis buffer. c. Lyse cells by sonication (50 % power, 5 s/5 s on/off, for a total on time of 10 min) on ice. d. Collect a 10 mL aliquot of the lysate (total lysate, TL) in a 1.5 mL microcentrifuge tube for SDS-PAGE analysis; add the appropriate amount of loading dye (usually 5 mL or what is recommended by manufacturer) and freeze at À20 C. e. Centrifuge the lysate for 30 min at 17000 3 g at 4 C to separate the soluble extract from the insoluble proteins and cell debris: collect the soluble extract. Collect 10 mL aliquots each of the soluble extract (S) and the insoluble pellet (I) in 1.5 mL microcentrifuge tubes for SDS-PAGE analysis, add the loading dye to each tube, and freeze at À20 C. Discard the remaining insoluble portion according to standard biosafety protocols. f. Filter the soluble extract using 0.45 mm syringe filters and store at 4 C.
Alternatives: Lysis can also be carried out by using a French press (SLM Aminco). The harvested cells can be resuspended in ca. 25 mL of lysis buffer and homogenized by using a Potter-Elvehjem tissue grinder to obtain a homogeneous cell suspension and to disrupt any solid salt precipitate possibly formed during cellular growth. The resuspended cells are lysed performing three lysis cycles using the French press at 20,000 psi (1 psi = 6.9 kPa).
CRITICAL: While lysing, both procedures cause an abrupt temperature increase in the cell suspension. It is highly recommended to keep the lysate on ice and to use a container with good thermal conductivity during the procedure to prevent thermal protein denaturation that would negatively affect final protein yield.

Immobilized metal affinity chromatography (IMAC) (Day 3).
Note: The expressed SARS-CoV-2-3CL pro contains a His 6 -SUMO tag at its N-terminus that allows for protein purification using immobilized nickel affinity chromatography carried out using an Ä KTAprime system with a 5 mL HiTrap Chelating HP column (Cytiva) working at a flow rate of 4 mL min À1 .
Note: Due to the high amount of His 6 -SUMO-3CL pro expressed from 1 L of cell culture, it is recommended to split the soluble fraction and perform the IMAC in two steps to prevent column overloading and possible loss of protein. Two or more columns placed in series can also be used to achieve high amounts of protein recovery and speed up the purification procedure. e. Collect a 10 mL aliquot of FT in a 1.5 mL microcentrifuge tube for SDS-PAGE analysis, add the loading dye, and freeze at À20 C. Store the rest of FT at 4 C. f. Elute the bound proteins by increasing the imidazole concentration by setting a linear gradient from 0 to 100 % of elution buffer in ten CVs and collecting eluate in 2 mL fractions. A representative chromatogram is shown in Figure 3A. g. Collect a 10 mL aliquot of each fraction in 1.5 mL microcentrifuge tubes for SDS-PAGE analysis, add the loading dye and freeze at À20 C. h. Once the elution is complete, wash the column with five CVs of elution buffer, MilliQ water, and 20 % v/v ethanol each to return the column in storing conditions. Store the column at 4 C. i. Analyze the total lysate (TL), the soluble extract (S), as well as the FT and the fractions eluted from the IMAC, for protein presence and purity by running an SDS-PAGE loading 5 mL of the samples previously collected and boiled for 5 min at 90 C (a representative protein gel is shown in Figure 3B). j. Pool the fractions containing His 6 -SUMO-3CL pro and store at 4 C. 4. Cleavage of the His 6 -SUMO tag by SUMO protease and buffer exchange (Day 3).
Note: The proteolytic cleavage of the N-terminal His 6 -SUMO tag is performed by the addition of His 8 -MBP-SUMO pro to the expressed His 6 -SUMO-3CL pro . General protocols for SUMO protease cleavage recommend carrying out cleavage in the presence of imidazole concentrations lower than 150 mM to prevent adverse effects of imidazole on the activity of SUMO protease. His 6 -SUMO-3CL pro elutes at ca. 180 mM imidazole concentration from the IMAC, therefore a buffer exchange may be useful to decrease the concentration of imidazole in the protein solution prior to treating with SUMO protease. However, uncontrolled cleavage of His 6 -SUMO tag from His 6 -SUMO-3CL pro , probably by trace amounts of native E. coli proteases, has been observed prior to the addition of SUMO protease. This can be minimized by carrying out cleavage in imidazole immediately after purifying the His 6 -SUMO-3CL pro fusion, with minimal to no negative effects on the production of pure and native 3CL pro (as described in this protocol). Nevertheless, if SUMO protease activity issues are observed, a buffer exchange into the Loading / Washing Buffer can be carried out before treatment with SUMO protease by performing points c-e before points a and b of the following procedure. Buffer exchange can be carried out using an Ä KTAprime system and a HiPrep 26/10 Desalting column (Cytiva) working at a flow rate of 10 mL min À1 , or by dialysis. In any case, buffer exchange is mandatory to decrease the concentration of imidazole in the protein prior to the following protocol for removal of the cleaved tag and the SUMO protease from the native 3CL pro by reverse IMAC.
Note: Incubation times for complete cleavage of N-terminal His 6 -SUMO tag by SUMO protease may vary depending on protease activity. As described in the before you begin section, the optimal ratio and cleavage temperature (4 or 26 C) should be empirically determined by performing cleavage assays on small-scale samples. In this protocol a 1:100 (w/w) ratio of SUMO protease generally provides > 99 % cleavage after 3 h when carried out in a pH 8.0 buffer containing reducing agent (e.g. the Elution Buffer) at 26 C.
a. Collect a 10 mL aliquot of the pre-cleaved His 6 -SUMO-3CL pro in a 1.5 mL microcentrifuge tube for SDS-PAGE analysis, add the loading dye, and freeze at À20 C. Alternatives: Cleavage of His 6 -SUMO tag from 3CL pro and buffer exchange can also be carried simultaneously by dialyzing the protein eluted from the IMAC in the presence of a 1:100 (w/w) ratio SUMO protease, for 12-16 h at 4 C, against Loading / Washing Buffer prepared without imidazole (i.e., 20 mM Tris pH 8.0, 300 mM NaCl, 1 mM DTT).

Removal of the cleaved tag and SUMO protease from cleaved 3CL pro solution (Day 4).
Note: The cleaved His 6 -SUMO tag and His 8 -MBP-SUMO pro both present N-terminal His-tags allowing for separation from the native 3CL pro through an additional step of IMAC. Cleaved 3CL pro will not interact with the column resin and will therefore be collected in the flowthrough, while His 6 -SUMO tag and His 8 -MBP-SUMO pro will bind to the column and will be eluted at a higher imidazole concentration. Separation is carried out using an Ä KTAprime system and a 5 mL HiTrap Chelating HP column (Cytiva) loaded with Ni 2+ , working at a flow rate of 4 mL min À1 .
a. Wash the 5 mL HiTrap Chelating HP column (Cytiva) used the day before with five CVs of MilliQ water and equilibrate it with five CVs of Loading / Washing Buffer. b. Load the protein solution onto the column using a superloop (Cytiva) or pump A (while using Loading / Washing Buffer).
c. Wash the column with Loading / Washing Buffer while monitoring the absorbance at 280 nm (Abs 280 ) to recover the cleaved, native 3CL pro in the flowthrough. Store the protein solution at 4 C. d. Collect a 10 mL aliquot of protein solution in a 1.5 mL microcentrifuge tube for SDS-PAGE analysis, add the loading dye, and freeze at À20 C. e. Elute the bound proteins remaining on the column using five CVs of 100 % Elution Buffer until the Abs 280 baseline returns to zero. f. Collect a 10 mL aliquot of the eluted proteins in a 1.5 mL microcentrifuge tube for SDS-PAGE analysis, add the loading dye and freeze at À20 C. g. Wash the column with five CVs of MilliQ water and five CVs of 20 % v/v ethanol to return the column in storing conditions. Store the column at 4 C.
Alternatives: To minimize cleavage of His 6 -SUMO tag from 3CL pro by native E. coli proteases, an on-column cleavage of His 6 -SUMO tag can be performed directly on the 5 mL Hi-Trap Chelating HP column prior to eluting with imidazole. This useful shortcut allows to perform IMAC purification, cleavage of His 6 -SUMO tag from 3CL pro , and removal of cleaved His 6 -SUMO tag and His 8 -MBP-SUMO pro from native 3CL pro in one step. In the on-column cleavage protocol, follow points a-e of step 3 (Immobilized metal affinity chromatography).
Then add 1-2 mL of 1 mg mL À1 His 8 -MBP-SUMO-pro (1 mL is sufficient for lower yields, e. g., batches fermented in minimal medium, but 2 mL may be needed for higher yield batches). Run one CV of Loading / Washing Buffer to fully load the protease onto the column and allow cleavage by storing the column 12-16 h at 4 C. The next morning, elute the cleaved 3CL pro with Loading / Washing Buffer and use the Elution Buffer to elute any remaining uncleaved His 6 -SUMO-3CL pro , the His 8 -MBP-SUMO pro , and the cleaved His 6 -SUMO tag. In this protocol, the target product is also not exposed to high concentrations of imidazole.

Size exclusion chromatography (SEC) (Day 4).
Note: A final size exclusion chromatographic (SEC) step provides a fully monodisperse and pure 3CL pro sample in a buffer suitable for downstream structural and biochemical studies. This step is carried out using an Ä KTAprime system and a HiLoadâ 16/60 Superdexâ 75 column (Cytiva), working at a flow rate of 1 mL min À1 .
a. Concentrate the protein solution to a final volume % 5 mL using an Amicon Ultra Centrifugal Filter Unit (MWCO 10,000 Da) (Merck Millipore).
CRITICAL: Although not strictly necessary, this concentration step is highly recommended to expedite the following SEC. During this step it is important to monitor the concentration of the protein solution (by measuring the absorbance at 280 nm and considering ε 280 = 32,890 M À1 cm À1 ), which should not exceed 0.8 mM to avoid possible precipitation. If the desired volume of 5 mL is not achievable, split the protein solution into two or more aliquots to be independently treated in the next purification step.
b. Wash the column with one CV of MilliQ water and equilibrate it with one CV of SEC buffer. c. Load up to 5 mL of protein solution using a sample loop of the appropriate volume and carry out the protein elution using SEC buffer (a representative chromatogram is shown in Figure 4A), collecting the eluate in 1 mL fractions and store at 4 C. d. Collect a 10 mL aliquot of each fraction in 1.5 mL microcentrifuge tubes for SDS-PAGE analysis, add the loading dye, and freeze at À20 C. e. Repeat points c -d for each 5 mL of the remaining protein solution. g. Analyze all collected fractions for protein presence and purity by running an SDS-PAGE gel loading 5 mL of the samples previously collected and boiled for 5 min at 90 C. Pool the fractions containing native 3CL pro . A representative SDS-PAGE is shown in Figure 4B. h. Native 3CL pro can be concentrated up to 25 mg mL À1 (corresponding to 0.8 mM monomer). At these concentrations, native 3CL pro can be flash-frozen in liquid nitrogen and stored for up to one month at À80 C.
Note: SEC buffer here reported is one among the several buffers in which native 3CL pro can be stored. Buffers most compatible with other downstream studies can be chosen as well.
Alternatives: In our experience, the cleaved His 6 -SUMO tag and the His 8 -MBP-SUMO pro can also be effectively separated from the native 3CL pro directly through the SEC procedure, rather than using the HiTrap column. To pursue this alternative, follow the purification procedure until point 4b (Cleavage of the His 6 -SUMO tag by SUMO protease), then skip step 4 points c -e (Buffer exchange) and step 5, and go directly to step 6.

Characterization of SARS-CoV-2 3CL pro by mass spectrometry
Timing: 3 h 7. Sample separation by HPLC. a. Dilute a 1 mg mL À1 solution of 3CL pro 1:10 (v/v) in MilliQ water. b. Load the diluted protein sample onto a Jupiter C4 column (Phenomenex) working at a flow rate of 0.4 mL min À1 using mobile phases A (H 2 O/formic acid; 100/0.1; v/v) and B (acetonitrile/formic acid; 100/0.1; v/v) to perform a gradient to enhance the separation of the eluting species. 8. Analyze the eluted species using an ESI-Q-TOF spectrometer (Xevo, Waters) using the software provided with the instrument.
Note: The resulting deconvoluted spectrum shows a single signal corresponding to an experimental molar mass of 33,796 Da ( Figure 5). The experimental value perfectly matches that estimated by the amino acid protein sequence (UniProt entry P0DTD1, residues 3264-3569) and establishes that the current protocol for the expression and purification of SARS-CoV-2 3CL pro is a reliable procedure to obtain large quantities of native 3CL pro . Note: 3CL pro inhibition properties are characterized by performing FRET assays using peptide substrate KTSAVLQ/SGFRKME (designed with the 3CL pro cleavage site Q/S) labeled with a DABCYL and EDANS FRET pair (fluorophore/quencher) on N and C termini, respectively (DABCYL-KTSAVLQ/SGFRKME-EDANS) (GenScript). Excitation at 360 nm excites the donor EDANS which transfers its energy non-radiatively due to the close proximity of the acceptor (quencher), DABCYL. Upon enzymatic cleavage of the labeled FRET peptide by 3CL pro , energy transfer no longer occurs between the fluorophore and quencher, resulting in an increase in fluorescence that can be detected at the emission maximum of the fluorophore EDANS (460 nm). This rate of increase in fluorescence is directly proportional to the enzyme activity.
FRET assays are carried out at increasing concentrations of potential 3CL pro inhibitors to screen candidate molecules and evaluate their IC 50 values (i.e., the inhibitor concentration providing a 50 % decrease of enzyme activity).
FRET assays are carried out using black opaque 96-well microplates (Corning) containing a final volume of 100 mL per well. Figures 6 and 7 provide the detailed amounts of reagents placed in each well. Experiments should be run in triplicate and results are monitored and analyzed using a TECAN Infinite M1000 microplate reader and Magellanä software (or comparable multi-well fluorimeter).

Instrument protocol setup:
Use the Magellanä software of TECAN Infinite M1000 microplate reader with the following parameters: (i) Excitation wavelength: 360 nm; (ii) Emission wavelength: 460 nm. Fluorescence intensity is recorded every 3 s for a total of 15-60 min at room temperature (20-25 C).

Calibration curve for fluorescence-to-activity conversion.
Note: Raw data collected during the FRET experiments indicate fluorescence intensity due to substrate proteolysis and are reported in terms of Relative Fluorescence Units (RFU). Preparation of a calibration curve for the conversion of the fluorescence data to cleaved peptide concentration (mM) using 0.10 to 20 mM EDANS is described below: Note: The resulting best-fit equation will be used to interpolate experimental fluorescence data obtained in the following experiment and convert from RFU per second to substrate hydrolysis rates (mM s À1 ).
Alternatives: A calibration curve can also be set up by incubating 200 nM 3CL pro with increasing concentrations of FRET substrate (0.5-50 mM) and monitoring the reaction until the fluorescence signal reaches plateaus for all concentrations, thus indicating that the total amount of the FRET substrate has been cleaved by 3CL pro . Final fluorescence values can be plotted as a function of the FRET substrate concentration and fit linearly to generate a calibration curve as described in step 10.
Note: For certain FRET studies it is necessary to calibrate the fluorimeter for inner-filter effects arising from UV absorbance by the substrate (or inhibitor) at high concentrations. 20 However, at the concentrations of substrate described in this protocol, no inner filter effect corrections are required.
11. 3CL pro , FRET substrate and inhibitor preparation: a. Prepare a 10 mM peptide substrate stock solution in DMSO, and store it as 60 mL aliquots at À20 C. b. Prepare 5 mM peptide substrate samples from the 10 mM stock by diluting 35 mL with an equal volume of FRET buffer. c. Prepare inhibitor stocks dissolving them in DMSO and diluting to 10.0, 1.0, and 0.1 mM concentrations in DMSO. d. Gently thaw 30 mL of purified 3CL pro on ice. Estimate the protein concentration using Lambert-Beer equation, with ε 280 = 32,890 A.U. M À1 cm À1 , and dilute the 3CL pro sample to a final concentration of 10 mM using FRET Buffer. 12. Setup of the reaction mixtures in the 96-well microplates:  i. FRET buffer as described in Figures 7A and 7B (90 mL of FRET Buffer will be added to every well except for column 2, where a negative control experiment in the absence of enzyme will be set up using 94 mL of FRET Buffer). ii. 4 mL of 10 mM 3CL pro stock solution (resulting in 400 nM 3CL pro final concentration) except for the negative control experiment in column 2. iii. 0-2.0 mL inhibitor, corresponding to 0-200 mM concentration (refer to Figures 7A and 7B for details). iv. Add the appropriate amount of DMSO to maintain the DMSO concentration constant in all wells. v. Let the plate equilibrate for 10-15 min at room temperature (20-25 C). vi. Initiate the reaction by adding 4 mL of 5 mM peptide substrate stock (resulting in 200 mM substrate final concentration), and mix by pipetting up and down 2-3 times.
Note: The enzymatic reaction begins as soon as the substrate is added to each well. Carefully pipette the substrate into each well, moving as quickly as possible to initiate the FRET data collection (below) with minimal dead time.
b. Place the 96-well microplate in the Infinite M1000 plate reader (TECAN) and press START on the Magellan software immediately to begin data collection. c. Let the reactions run for 10-20 min to measure initial enzyme velocities.  Note: Inhibitors vary in their solubility. Therefore, the assay may be adjusted to use final concentrations of DMSO even lower than 1 % for appropriately soluble substrates and inhibitors. 13. Data export and analysis.
a. Export the data from Magellan in Microsoft Excel .xls or in .csv format. b. Analyze the data using GraphPad or other kinetic analysis software: i. Plot the fluorescence intensity vs. time data ( Figure 7C) for each inhibitor concentration.
ii. Measure the slope of the linear portion of each curve (usually within the first 5 min of reaction) by performing a linear fit on that region.
Note: Each obtained slope value corresponds to the initial velocity in the presence of that inhibitor concentration (V i ). The slope determined for the positive control measurements in column 1 ( Figure 7B), carried out in the absence of inhibitor, corresponds to the initial velocity of the non-inhibited 3CL pro (V 0 ).
iii. Calculate the average G standard deviation values for the triplicate measurements carried out at each inhibitor concentration to obtain, for each concentration, an averaged V i (or V 0 ) value. iv. Using the averaged values, calculate residual activity (%) at each inhibitor concentration using the formula v. Plot residual activity (%) data as a function of increasing inhibitor concentration and fit them by using the following equation: Residual activity ð%Þ = 100 1+x=IC 50 (Equation 2) vi. Transform the x-axis visualization on a Log 10 scale to show the plotted experimental data and resulting fit following the typical sigmoidal behavior ( Figure 7D).
Note: The IC 50 value estimated by Equation 2 corresponds to the inhibitor concentration at which residual activity is 50 %.
Alternatives: This protocol can also be adopted as a method for preliminary screening of a panel of molecules as potential inhibitors by using a single concentration (e.g., 20 mM) of each.  Alternatives: Alternative NMR software for processing include Bruker TopSpin 4.2.0, and earlier releases, the CcpNmr software suite, or software available in NMRBox software collection. 21,22 An extensive review of NMR data processing is presented in Stern and Hoch (1996). 23 Sequence-specific resonance assignments for SARS-CoV-2 3CL pro are available in the BMRB entry 50780 24 and can be used to validate the spectrum and to label the observed peaks following the nearest neighbor criterium.

Crystallization and structural determination of SARS-CoV-2 3CL pro
Timing: Approximately 2 weeks Note: SARS-CoV-2 3CL pro can be crystallized at 3-8 mg mL À1 through vapor diffusion technique (hanging drop method) using a 20-60 mM ammonium acetate buffer at pH 7.0 and 20-30 % PEG4000 as a precipitant. This protocol reports a representative crystallization procedure using a 5 mg mL À1 sample. 16. Crystallization of SARS-CoV-2 3CL pro .
a. Prepare the crystallization cocktail by filling each well (0.5 mL) of the EasyXtal 15-well crystallization plate (Quiagen) with the correct volumes of 1 M ammonium acetate at pH 7.0 (AMA 1 M), 50 % PEG4000 (PEG 50 %), and MilliQ water according to the scheme in Table 1, as shown in Figure 9A.
Note: Due to different viscosities of the three solutions added, it is recommended to mix the content of each well using a micropipette.
b. Gently thaw 50 mL of purified 5 mg mL À1 3CL pro on ice. c. Place a screw-in crystallization support upside down and pipette 2 mL of 3CL pro solution at its center. Alternatives: The same experimental setup used to crystallize native 3CL pro can be also successfully followed, after minor changes, to crystallize the protein in the presence of potential inhibitors by using either co-crystallization or crystal soaking. In the first case, appropriate amounts of test molecule should be added in the crystallization cocktail (point 16a) prior to drop deposition, in order to obtain a co-crystallization drop containing both the protein and the molecule. To perform the crystal soaking, procedure described at point 17c (see below), the protocol should be modified by exposing a crystal of native 3CL pro in a cryo-protectant droplet that also contains the appropriate amount of ligand. During soaking the ligand may diffuse into the preformed crystal through solvent channels and bind to the protein. Exposure time of the protein to the ligand needs to be optimized to achieve complete binding (usually from a few minutes to 12-16 h). CRITICAL: The liquid contained in the drops tends to evaporate quickly. This phenomenon also causes the disruption of the crystal order, which in turn negatively affects data quality. Crystal fishing and flash-cooling procedures must be therefore carried out as quickly as possible. If additional time is needed between two consecutive crystal fishing steps, place the drop support back onto the corresponding well to minimize drop drying.
Note: Diffraction data used for the structural determination of the X-ray crystal structure of native SARS-CoV-2 3CL pro were collected on one crystal at 100 K using synchrotron X-ray Disallowed regions (%) 0.4 a Highest resolution bin in parentheses; b R sym = P hkl P j I j À CID = P hkl P j I j , where I is the intensity of a reflection, and CID is the mean intensity of all symmetry related reflections j; c R p:i:m: = P hkl f½1=ðN À 1Þ 1=2 P j I j À CID g= P hkl P j I j , where I is the intensity of a reflection, and CID is the mean intensity of all symmetry related reflections j, and N is the multiplicity 28272827282828282726252626272727272727272625242322222118 ; d Taken from REFMAC; R free is calculated using 5% of the total reflections that were randomly selected and excluded from refinement; e DPI = R factor $D max $compl À 1 = 3 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi N atoms ðN refl À N params Þ s , where N atoms is the number of the atoms included in the refinement, N refl is the number of the reflections included in the refinement, D max is the maximum resolution of reflections included in the refinement, compl is the completeness of the observed data, and for isotropic refinement, N params z 4N atoms radiation at the EMBL P13 beamline of the Petra III storage ring, c/o DESY, Hamburg (Germany). 25 a. Record diffraction data using the collection strategy suggested by the MXCuBE software. 13,14 b. Process X-ray diffraction images by using XDS. 15 c. Scale and reduce processed data by using AIMLESS, 17 included in the suite CCP4. 16 d. Carry out structural refinement and model building using the REFMAC5 software 18 and COOT, 26 respectively, both included in the suite CCP4. 16 Use the structure factor amplitudes obtained by AIMLESS as experimental data and the PDB entry 7ALH as a starting model until refinement convergence. A representative picture of the final X-ray crystal structure of 3CL pro obtained in this work (and deposited in the Protein Data Bank under PDB code 8CDC) is shown in Figure 10. Data collection, processing and refinement statistics are reported in Table 2.
Alternatives: Other data collection facilities may be used. Other software can be used for analyzing the crystallographic data, such as PHENIX. 27 Note: Crystals grown in experimental conditions described belong to space group C121, as does the starting model selected for the refinement (PDB code 7ALH). If the space groups of  experimental data and the starting model do not match, a molecular replacement procedure must be carried out to obtain initial crystallographic phases to be used in the following refinement and model building steps.

Sample quality control
In addition to crystallography and NMR data, if available, sample quality control includes SDS-PAGE, MALDI-TOF mass spectrometry, and specific enzyme activity measurements. The expected specific activity is 55 Units / mmole of enzyme, where a Unit is 1.0 mmol substrate cleaved / min, assayed using the FRET buffer conditions, as described in ''materials and equipment'' section, using 0.4 mM 3CL pro , 200 mM DABCYL-KTSAVLQ/SGFRKME-EDANS substrate at 25 C.

EXPECTED OUTCOMES
This paper provides a detailed protocol for the expression, purification, and production of tens-ofmilligram quantities of highly pure (> 99 %) SARS-CoV-2 3CL pro with native N and C termini and in a biochemically-active form, as demonstrated by the bio-structural characterization and activity assays reported. Yields of pure SARS CoV-2 3CL pro obtained, as high at $ 120 mg / mL, depend on the medium used for fermentation (Table 3).
3CL pro samples produced by this protocol are stable in NMR measurements for up to 1 week at room temperature and provide excellent HSQC-TROSY spectra suitable for structure, dynamic, ligand binding studies. Moreover, the set-up crystallization protocol provides high quality 3CL pro X-ray crystal structures at resolution better than 2 Å , thus suitable for biophysical and drug discovery research using X-ray crystallography. In the paper a standardized assay for estimating IC 50 for new potential 3CL pro inhibitors is also presented, thus complementing structural information with biochemical insights of catalytic and inhibition mechanism of this target.

LIMITATIONS
A percentage of the obtained 3CL pro crystals ($ 20 %) do not diffract at high resolution. The protocol section that describes the characterization of enzyme inhibition by FRET assays assumes solubility of the screening molecules in 4 % DMSO. For potent inhibitors which can be studied at lower concentrations, lower DMSO concentrations may be used if solubility allows. Each potential inhibitor should be evaluated for solubility prior to setting up the assay. Moreover, IC 50 obtained from the biochemical assays is a preliminary measurement of inhibitor potency. A more accurate investigation of the inhibition should be provided by determining thermodynamic and kinetic parameters for the inhibition mechanism.
TROUBLESHOOTING Problem 1 Following the 12-16 h cell growth on LB Petri dishes, no colonies are visible.

Potential solution
After checking each step of the transformation procedure was carried out correctly (e.g., correct antibiotic, plasmid concentration, duration of heat shock), the concentration of the cell suspension can be increased via centrifugation. At the end of section 13f in the ''before you begin'' section, centrifuge the cell suspension for 1 min at 2500 3 g and decant half of the supernatant. Resuspend the pellet in the remaining supernatant for a more concentrated cell culture.

Problem 2
During cell lysis (step 2. Cell lysis and soluble extract recovery), heat generated by sonication and French Press, can cause aggregation or misfolding of 3CL pro Potential solution Sonication should be done in short blasts, with the sample cooled in an ice bath in a container with good thermal conductivity. Lysis through French press should be also performed constantly maintaining the sample in an ice bath. Microfluidizer or homogenizer can be used in place of the aforementioned techniques, though lysis by these methods may be less complete resulting in lower final yields.

Problem 3
The expressed His 6 -SUMO-3CL pro undergoes a first step of purification through an immobilized nickel affinity chromatography (IMAC) (step 3). Due to the high amount of protein expressed from 1 L of cell culture and a column capacity of usually 10 mg mL À1 of resin (indicating that a 5 mL column will efficiently bind up to 50 mg of protein), column overloading issues can be encountered. Such an event is clearly detectable by running an SDS-PAGE of the flow-through, which would show large amounts of unbound His 6 -SUMO-3CL pro .

Potential solution
It is strongly recommended to split the soluble lysate in two or more aliquots and perform the IMAC in successive steps. As an alternative, two or more columns placed in series can also be used to achieve high amounts of protein recovery and speed up the purification procedure.

Problem 4
Uncontrolled cleavage of His 6 -SUMO tag from His 6 -SUMO-3CL pro probably by trace amounts of native E. coli proteases has been observed at the end of step 3 (IMAC), just before the addition of His 8 -MBP-SUMO pro .

Potential solution
Uncontrolled cleavage can be minimized by carrying out cleavage in imidazole immediately after purifying the His 6 -SUMO-3CL pro , with low to no negative effects on the production of pure and native 3CL pro , or by on-column cleavage, as suggested by this protocol.

Problem 5
The on-column cleavage protocol set up to perform IMAC purification, SUMO protease cleavage of His 6 -SUMO tag from 3CL pro , and removal of cleaved His 6 -SUMO tag and His 8 -MBP-SUMO pro from native 3CL pro in one step is more efficient and quicker. However, in some cases subsequent elution of the proteins reveals incomplete cleavage of the His 6 -SUMO-3CL pro , resulting in reduced final yields.
Potential solution His 6 -SUMO-3CL pro fusion that elutes from the column can be dialyzed to remove imidazole and undergo a second on-column cleavage procedure.

Problem 6
Incomplete cleavage of the His 6 -SUMO-3CL pro fusion is observed (i.e., full target fusion is visible in the final elution from the IMAC).

Potential solution
Collect the elution fractions containing the His 6 -SUMO-3CL pro fusion, perform an 12-16 h dialysis/ buffer exchange and simultaneous cleavage in the presence of H 8 -MBP-SUMO pro and repeat an IMAC step by collecting the flow-through.

Problem 7
No increasing fluorescence signal is observed after combining enzyme and substrate during the FRET Assay control (described in step 12).
Potential solution 3CL pro is very sensitive to oxidation and can lose enzymatic activity due to oxidation of the active site cysteine even though reducing agents are present. During purification it is advantageous to work quickly and at 4 C as much as possible to minimize this effect. Samples can also be protected from O 2 by storing them under nitrogen or argon gas.

Problem 8
Quality of 15 N HSQC spectrum is not optimal.

Potential solution
Prepare a 2 H, 15 N -enriched sample to obtain a higher quality 15 N HSQC spectrum.

Problem 9
Reservoir solutions for crystallization trials includes mixing of ammonium acetate, 50 % PEG4000, and water, which have different viscosity and may prevent the final solution to be homogeneous.

Potential solution
Carefully mix the reservoir solution present in each well using a micropipette.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, G.T. Montelione, monteg3@rpi.edu.