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Sep 2021

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Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein
与 SARS-CoV-2 刺突蛋白受体结合域复合的纳米抗体的产生和结晶   

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Abstract

The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural analysis of VHH-RBD complexes by X-ray crystallography enables VHH epitopes to be precisely mapped, and the effect of variant mutations to be interpreted and predicted. Key to this is a protocol for the reproducible production and crystallization of the VHH-RBD complexes. Based on our experience, we describe a workflow for expressing and purifying the proteins, and the screening conditions for generating diffraction quality crystals of VHH-RBD complexes. Production and crystallization of protein complexes takes approximately twelve days, from construction of vectors to harvesting and freezing crystals for data collection.

Keywords: SARS CoV-2 (SARS-CoV-2), Nanobodies (纳米抗体), Receptor Binding Domain (受体结合域), Protein purification (蛋白质纯化), Crystallization (结晶)

Background

Camelids (llamas, alpacas, and camels) produce a unique type of heavy chain only antibody that comprises variable domains (VHH) linked to Fc constant domains (CH2 and CH3). The VHHs can be produced as single domain antigen-binding proteins or nanobodies (Muyldermans, 2013), with wide applications in the life sciences (Pleiner et al., 2015; Jovcevska and Muyldermans, 2020). In response to the COVID-19 pandemic (Dhama et al., 2020), we and many other groups have developed VHH reagents that bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein that block binding to ACE-2 at the cell surface, which is the primary interaction that leads to cell infection (Xiang et al., 2020; Hanke et al., 2020; Schoof et al., 2020; Koenig et al., 2021; Huo et al., 2020, 2021). Multimeric versions of these VHHs, either as trimers or IgG Fc fusions, have been shown to neutralise SARS-CoV-2 both in vitro and in animal models of COVID-19 (Huo et al., 2021; Nambulli et al., 2021). Structural analysis of VHH-RBD complexes has revealed two regions where epitopes are clustered, one at or close to the ACE-2 binding surface (cluster 2), and one at the opposite side of the RBD (cluster 1) (Tang et al., 2021). Information about the VHH binding sites has enabled the effect of mutations in the spike protein to be interpreted and predicted. In our experience, forming RBD complexes with two VHH that bind to orthogonal sites has been necessary for the crystallization of some VHH-RBD complexes (Huo et al., 2021).


VHHs are routinely produced in the E. coli strain WK6 as hexahistidine tagged proteins, using Isopropyl β-D-1-thiogalactopyranoside (ITPG) inducible promoters, and the addition of a secretion signal (e.g., pelB or ompA), to enable recovery of the VHH from the periplasm following induced expression. Primary purification uses immobilised metal affinity chromatography (IMAC) with gel filtration added as a polishing step. Alternatively, VHHs can be directly purified from lysed cells though, in our experience, periplasmic extraction by osmotic shock gives higher protein yields.


As a glycosylated protein, the production of the RBD of the SARS-CoV-2 spike protein requires expression in higher eukaryotic cells for which both insect and mammalian cells have been used. An appropriate signal sequence is added to the N-terminus to direct product to the cell media, and a hexahistidine tag added to the C-terminus for purification by IMAC. The N-glycosylation of the RBD (Asn331 and Asn343) introduces chemical heterogeneity, particularly if produced in mammalian cells, which generally inhibits crystallization (Chang et al., 2007 ). By growing cells in the presence of the mannosidase inhibitor kifunensine, N-glycosylation can be arrested at a high mannose state (GlcNAc2Man9 glycoforms). These can subsequently be trimmed back to single N-acetylglucosamine residues, by treatment with endoglycosidase F1 or H (Chang et al., 2007). Based on past experience (Nettleship et al., 2013), this is most efficiently carried out prior to purification of the VHH-RBD complex.


An issue for the purification of secreted glycoproteins from cell culture media by IMAC is that some media components displace the His-tagged protein-of-interest during the chromatography step, significantly reducing the yield. Here, we have used an automated method of affinity purification by IMAC and gel filtration, which involves loading the sample onto the Ni-NTA column in batches, with a column washing step between each batch (Nettleship et al., 2009).


We have reported the crystallization of a number of VHH-RBD complexes using commercially available screens, in 96-well format, and low sample volumes of 1–200 nL. Diffraction data were collected from the primary crystal hits using synchrotron X-rays, and structures solved by molecular replacement (Huo et al., 2020, 2021). Key to achieving good quality crystals was the preparation of the proteins. Thus, in this article, we describe our optimised workflow for producing VHH-RBD complexes and their crystallization for analysis by X-ray crystallography.

Materials and Reagents

  1. Production of a receptor binding domain

    1. Vector construction

      1. pOPINTTG vector digested with KpnI/PmeI (Figure 1A)

      2. Human codon optimised synthetic RBD gene (encoding amino acids 330–352) with 15-bp extensions overlapping with pOPINTTG in-fusion entry sites (lower case):

        5’gcgtagctgaaaccggcCCGAATATCACAAATCTTTGTCCTTTCGGAGAAGTATTCAACGCAACTCGCTTCGCATCAGTATATGCCTGGAACCGCAAACGAATTTCTAATTGTGTCGCCGACTACTCTGTGCTTTACAACTCAGCATCATTTTCAACATTCAAATGCTATGGTGTCTCCCCTACGAAGTTGAACGATCTTTGTTTCACTAATGTCTACGCCGATTCCTTTGTTATTAGGGGGGACGAGGTTCGCCAGATCGCGCCGGGCCAAACGGGCAAGATAGCTGACTATAATTATAAGCTGCCAGACGACTTTACAGGCTGCGTTATCGCTTGGAATTCAAACAATCTTGATAGCAAAGTGGGTGGTAATTATAACTACCTCTACAGACTGTTTCGCAAGTCTAATCTGAAGCCTTTCGAGCGGGACATCTCTACTGAGATCTATCAAGCTGGTTCAACCCCCTGCAATGGCGTCGAGGGTTTTAACTGTTACTTCCCACTTCAGTCATACGGATTTCAACCAACTAATGGGGTTGGCTATCAGCCGTACCGCGTGGTCGTTCTTAGCTTTGAGCTGCTTCACGCCCCTGCAACGGTGTGCGGACCGAAAAAAAGTACAAAaaacatcaccatcac 3’

      3. ClonExpress II One Step Cloning kit (Vazyme, catalog number: C113-02)

      4. NucleoSpin 148® Gel and PCR Clean-up kit (MACHEREY-NAGEL, catalog number: 12303368)

      5. Stellar competent cells (Takara Bio, catalog number: 636766)

      6. LB medium (Sigma-Aldrich, catalog number: L3022)

      7. S.O.C. (Super Optimal broth with Catabolite repression) recovery medium (ThermoFisher Scientific, catalog number: 15544034)

      8. Ampicillin (Sigma-Aldrich, catalog number: A9393)

      9. LB-agar plates supplemented with 100 μg/mL ampicillin

      10. Plasmid miniprep kit (e.g., Qiagen, catalog number: 27104)

      11. Plasmid Plus Midi kit (e.g., Qiagen, catalog number: 12941)

      12. Sequencing primers: pTTfwd 5’ TCCACAGGTGTCCACTCC 3’

        pTTrev 5’ TCCTTTATTAGCCAGAGG 3’

    2. Expression in expi293TM cell

      1. 500 mL sterile baffled flasks with vented closure (ThermoFisher Scientific, catalog number: 4116-0500)

      2. CountessTM cell counting chamber slides (ThermoFisher Scientific, catalog number: C10228)

      3. Expi293TM cells (ThermoFisher Scientific, catalog number: A14527)

      4. Expi293TM expression medium (ThermoFisher Scientific, catalog number: A1435101)

      5. Trypan blue solution (ThermoFisher Scientific, catalog number: 15250061)

      6. Hanks' Balanced Salt Solution (HBSS) (10×) (ThermoFisher Scientific, catalog number: 14185052)

      7. Gibco OPTI-MEM reduced serum medium (ThermoFisher Scientific, catalog number: 31985062)

      8. Valproic acid (Sigma-Aldrich, catalog number: P4543)

      9. Glucose (45% solution) (see Recipes or from Sigma-Aldrich, catalog number: G8769)

      10. Sodium propionate (Sigma-Aldrich, catalog number: P1880)

      11. PEI MAX 40 K (Polysciences Inc., catalog number: 24,765-1)

      12. Kifunensine (Sigma-Aldrich, catalog number: K1140)

      13. Pre-cast SDS polyacrylamide gels (e.g., NuPAGETM 10%, Bis-Tris ThermoFisher Scientific, catalog number WG1201A)

      14. InstantBlue® Coomassie protein stain (Abcam, catalog number: ab119211)

    3. RBD purification

      1. 5 ml HisTrap_FF Ni-NTA columns (Cytiva, catalog number: 1752860)

      2. SD75 16/600 size exclusion column (Cytiva, catalog number: 28989333)

      3. Imidazole (Sigma-Aldrich, catalog number: I2399)

      4. PBS, Phosphate Buffered Saline, 10× Solution (Fisher, catalog number: BP399-20)

      5. Wash buffer (see Recipes)

      6. Elution buffer (see Recipes)

      7. Gel filtration buffers (see Recipes)


  2. VHH production

    1. Vector construction

      1. pADL-23c vector cut with SfiI (Figure 1B) (New England Biolabs, catalog number: R0123S)

      2. Infusion primers:

        VHH Fwd primer 5’ GTTATTACTCGCGGCCCAGCCGGCCATGGCCC 3’

        VHH Rev primer 5’ GGTGATGGTGTTGGCCTTTATTAATGATGGTGGTGATGGTG 3’

      3. Proof reading polymerase (e.g., PhusionFlashTM high fidelity polymerase ThermoFisher Scientific, catalog number: F548S)

      4. pADL-23c sequencing primers: PhDseqFwd 5’ GCTTCCGGCTCGTATGTTG 3’

        PhDseqRev 5’ GTCGTCTTTCCAGACGTTAG 3’

      5. 2 mL Cryovials (e.g., ThermoFisher Scientific, catalog number: 5000-0020)

    2. E. coli expression

      1. WK6 cells Escherichia coli (Migula) Castellani and Chalmers (ATCC, catalog number: 47078)

      2. Terrific Broth (TB) Medium (see Recipes)

      3. Isopropyl β-D-1-thiogalactopyranoside, IPTG (Sigma-Aldrich, catalog number: I6758)

      4. Glucose

      5. Magnesium chloride

      6. Ampicillin

    3. VHH purification

      1. TES buffer (see Recipes)

      2. DNase I (Sigma-Aldrich, catalog number: D4263)

      3. Magnesium Chloride (Sigma-Aldrich, catalog number: M8266)

      4. Sucrose (Sigma-Aldrich, catalog number: S0389)



      Figure 1. Vector maps (A) pOPINTTGneo (B) pADL-23c


  3. Crystallization of VHH-RBD complexes

    1. Preparation of VHH-RBD complexes

      1. EndoH ( New England Biolabs, catalog number: P0702S)

      2. SD200 10/300 size exclusion column (Cytiva, catalog number: 28990944)

    2. Crystallization screening

      1. Swisssci Triple-Drop crystallization plates (Molecular Dimensions, catalog number: MD11-003-100)

      2. V well Microplate without lid, natural, polypropylene (Greiner, catalog number: 651201 96)

      3. VIEWsealTM plate sealer, transparent, non-piercable Greiner, catalog number: 676070)

      4. Pact premierTM crystallization screen condition: (Molecular Dimension, catalog number: MDSR-29)

      5. JCSG-plusTM crystallization screen condition: (Molecular Dimensions, catalog number: MDSR-37)

      6. SG1TM crystallization condition: (Molecular Dimensions, catalog number: MDSR-88)

    3. Cryopreservation of crystals

      1. Glycerol (Molecular Dimensions, catalog number: MD2-100-65)

      2. PEG 400 (Sigma-Aldrich, catalog number: 06855)

      3. Mounted Round LithoLoops (0.25 mm) (Molecular Dimensions, catalog number: MD7-137)

      4. Mounted Round LithoLoops (0.15 mm) (Molecular Dimensions catalog number: MD7-135)

      5. Standard Foam Dewar (Molecular Dimensions, catalog number: MD7-35)

      6. Uni-Puck 10 Pack ( Molecular Dimensions, catalog number: MD7-613)

      7. Cryotool set (Molecular Dimensions, catalog number: MD7-517)

      8. Dry Shipper (Molecular Dimensions, catalog number: MD7-21

Equipment

  1. Microvolume spectrophotometer (e.g., ThermoFisher Scientific, NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer: ND-ONE-W)

  2. CO2 orbital shaker (e.g., n-Biotek.com ANICELL incubator, catalog number: NB-206CXL/NB)

  3. Tabletop centrifuge suitable for 50 mL Falcon tubes (Sorvall Legend RT Plus)

  4. Cell Counter (e.g., ThermoFisher Scientific CountessTM 3 Automated Cell Counter)

  5. ÄKTA Xpress automated multi-step purification system

  6. Liquid handler (e.g., Hydra Dispenser by Art Robbins Instruments)

  7. Low volume dispenser for crystallization plates (e.g., SPT labtech mosquito® LV)

  8. Crystal plate imager (e.g., Formulatrix RockImager® system)

Procedure

  1. Production of receptor binding domains

    1. Vector construction

      1. For 10 μL of in-fusion reaction, mix 20 ng (1–3 μL) of synthetic gene, 100 ng (1–2 μL) of PmeI/NcoI cut pOPINTTG vector, 1 μL of Exnase II, and 2 μL of optimized buffer supplied with the cloning kit.

      2. Incubate the reaction at 37°C for 30 min, and then stop immediately, by adding 20 μL of ice-cold TE buffer.

      3. Use 5 μL of the resulting reaction mixture to transform 20 μL of Stellar competent cells, by incubating on ice for 30 min, then heat shocking at 42°C for 45 s.

      4. Add 400 μL of SOC media and incubate at 37°C for 45 min.

      5. Plate 100 μL of the cell and media mixture on LB-agar plates supplemented with 100 μg/mL ampicillin, and culture at 37°C overnight.

      6. Pick single colonies into 3 mL of LB containing 100 μg/mL ampicillin and incubate at 37°C overnight.

      7. Produce glycerol stocks, by adding 0.5 mL of 50% (v/v) sterile glycerol to 0.5 mL of overnight culture of cells in LB in a 2-mL cryovial, and freezing at -80°C.

      8. Prepare DNA from pelleted cells, and confirm correct clones by sequencing minipreps DNAs with pTTfwd and PTTrev primers.

      9. Use 50% glycerol stock to grow larger scale cultures for transfection-grade plasmid preparation.

      10. Purify transfection-grade plasmids (0.5–1 mg) from 150 mL of overnight LB culture, using QIAGEN Plasmid Plus Midi kit and, if required, store plasmids at -20°C in sterile 1.5-mL Eppendorf tubes.

      11. Measure the purity and concentration of DNA using NanoDrop spectrophotometer. Plasmid DNA used for transfections should be of high purity (see Note 1).

      12. The concentration and purity of DNA is calculated automatically by the instrument using the following equations:

        Concentration: DNA (µg/mL) = (A260 reading – A320 reading) × dilution factor × 50 µg/mL

        Purity: (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)

      13. Use 1 μg DNA per one million of transfected cells.

    2. Transfection protocol

      1. Maintain the suspension culture of Expi293TM cells for at least three passages after defrosting (passage numbers 3–30 can be used in experiments) in a humidified (80%) incubator, with 5–8% CO2, at 37°C, on an orbital shaker at 120 rpm in Gibco Expi293TM Express medium, at a cell density between 0.5 and 5.0 × 106 cells/mL. Use a 125-mL flask to maintain 30 mL of Expi293 cells.

      2. One day before transfection, seed Expi293TM cells at a cell density of 1 × 106 cells/mL. For each preparation of RBD, set up three cultures of 170 mL in 500-mL flasks.

      3. On the day of transfection, determine cell count and viability, and if cell count is between 2 × 106 –2.5 × 106/mL and at least 95% viable, proceed with transfection (see Note 2).

      4. For each 170-mL culture, mix 17 mL of OPTI-MEM media with 170 μg plasmid DNA and 918 μL of PEI Max 40kDa transfection reagent in a 50-mL Falcon tube.

      5. Mix thoroughly, incubate at room temperature (RT) for 10 min, and add gently (dropwise) to the Expi293TM cells (see Note 3).

      6. Add kifunensine from a stock concentration of 1 mg/mL (100 μL per 100 mL of culture volume), and return cells to incubate on the orbital shaker at 125 rpm, 5–8% CO2, 80% humidity and 37°C.

      7. After 16–18 h, add to each 170 mL culture, 2,890 μL of valproic acid, 1,100 μL of sodium propionate, and 3,400 μL of glucose. Return the culture to the incubator (see Note 4).

      8. On day 5 after transfection, determine cell count and viability.

      9. Harvest media, and spin in a 0.5-L centrifuge bottle at 6,000 × g for 20 min. Filter sterilise using a 0.45 μm 0.5-L bottle top filter.

    3. Purification

      The IMAC-SEC purification protocol is for an ÄKTA Xpress platform using a programme described in Nettleship et al. (2009) . The same workflow can be implemented on other purification systems with automated peak detection. A transcript of the Unicorn programme is provided as an Appendix.

      1. Equilibrate the gel filtration column using the “Gel Filtration Equilibration” programme and the Gel filtration buffer (20 mM Tris, pH 7.5, 200 mM NaCl).

      2. Follow the method “Mammalian Prepping System” to set up the system. It will pump wash A1 and A2, and clean the inlets. It will then ask you to screw the 5-mL column into position 1, and will equilibrate the column.

      3. Add an equal volume of PBS to the filtered cell supernatant, and adjust the pH to 7.4 with NaOH.

      4. Once the column is plugged in and the sample is ready to be loaded, slowly remove the A2 line from the buffer (by small movements to avoid bubbles in the line), and insert it into the sample. Make sure all the lines are at the bottom of the bottles.

      5. Leave the protocol to run overnight.

      6. Using the trace from the ÄKTA, select the correct fractions containing the protein of interest. Mix 10 μL of protein solution with 10 μL of sample buffer, and heat at 95°C for 5 min. Run an SDS-PAGE gel of the samples, and stain it using Instant Blue®.

      7. Concentrate the protein, usually to 5 mg/mL, using a 10 kDa concentrator centrifuge at 2,500 × g and 4°C.

      8. Aliquot (e.g., 0.1 mL) and flash freeze the protein, by plunging tubes into liquid nitrogen.

      9. Store at -80°C.


  2. Production of VHHs

    1. Vector construction

      VHHs identified by screening M13 phage display libraries are re-expressed for protein production.

      1. Amplify VHH from source display vector using VHH Fwd primer and VHH Rev primers and the following PCR conditions:

        1) 98°C for 10 s

        2) 30 cycles of:

        98°C for 1 s

        60°C for 5 s

        72°C for 15 s

        3) 72°C for 2 min

        4) 4°C hold

      2. Purify amplified VHH DNA by agarose gel electrophoresis, and extract using the Nucleospin® kit according to the manufacturer’s instructions. Clone PCR product into the SfiI cut pADL-23c vector, as described for the cloning of the RBD.

      3. Verify clones, by sequencing with PhDseqFwd and PhDseqRev primers.

    2. Expression in E. coli

      1. Transform chemically competent WK6 cells as described above, for construction of the RBD expression vector.

      2. Pick a colony from the plate of transformed cells, and set up a pre-culture: 8 mL of TB supplemented with 100 μg/mL ampicillin, 2% glucose and 1 mM MgCl2.

      3. Set up the culture: 800 mL of TB medium in a 2-L flask supplemented with 100 μg/mL ampicillin, 0.1% glucose, and 1 mM MgCl2.

      4. Pre-warm the flask/medium to 37°C.

      5. Add pre-culture and grow culture on an orbital shaker at 225 rpm and 37°C.

      6. Add IPTG to a final concentration of 1 mM when OD600 reaches ~1.2 (usually takes around 3.5 h), and continue growing at 225 rpm and 28°C overnight.

      7. Pellet the cells at 2,500 × g at 4°C for 15 min.

    3. Purification of VHHs

      1. Add 15 mL of TES buffer to the cell pellet, and resuspend slowly in a bottle with a magnetic stirrer at 4°C overnight.

      2. The next day, add twice the volume of cold TES/4 buffer supplemented with 120U Kunitz DNaseI to the resuspended culture, and slowly stir for 2 h.

      3. Top up to 80-mL with TES/4 buffer.

      4. Spin down the pellet at 28,000 × g and 4°C for 30 min (in a 50-mL tube).

      5. Filter the supernatant through a 0.8 μm filter under vacuum.

      6. Dilute the supernatant with five volumes of PBS (pH 7.4), and mix well.

      7. Load the sample onto two 5-mL IMAC columns connected in series, at <2 mL/min.

      8. Wash the column with PBS (pH 7.4) containing 30 mM imidazole.

      9. Elute the sample with PBS containing 300 mM imidazole at 1 mL/min into a 96-well collection plate, collecting 1-mL fractions.

      10. Pool A280 peak fractions (approximately 7.5 mL) and run on a Superdex S75 16/600 in gel filtration buffer (50 mM Tris pH 7, 150 mM NaCl).

      11. Pool A280 peak protein fractions, and concentrate using a 5 kDa MWCO concentrator, usually to 15 mg/mL (see Note 5).

      12. Aliquot (e.g., 0.1 mL) and flash freeze the protein, by plunging tubes into liquid nitrogen.

      13. Store at -80°C.


  3. Crystallization of VHH-RBD complexes

    1. Preparation of VHH:RBD complexes

      1. Mix 5 mg of RBD (5 mg/mL) with 3 mg VHH (15 mg/mL) at a molar ratio RBD:VHH of 1:1.2, and incubate under agitation at 2 rpm in a cold room for 3 h (see Note 6).

      2. Incubate the RBD-VHH complex with 0.4 mg of EndoH glycosidase (1 mg/mL) under agitation at 2 rpm at room temperature overnight (see Note 7).

      3. Concentrate the mixture to 1 mL with a 5 kDa MWCO concentrator, and inject onto a Superdex 200 10/300 in gel filtration buffer (50 mM Tris pH 7, 150 mM NaCl).

      4. Monitor A280, pool peak fractions, and concentrate using 5 kDa MWCO concentrator to 20 mg/mL (see Figures 2 and 3).



      Figure 2. Purification of VHH-RBD complex on a SD200 20/300 size exclusion column (CV 23.56 mL).



      Figure 3. SDS-Page of RBD (lane 1), VHH (lane 2), and fractions from gel filtration of RBD-VHH complexes, following treatment with endoglycosidase HH (lanes 3–12).


    2. Crystallization screening

      1. Dispense 25 μL of crystallization solution from 96-well masterblock into the reservoir well of Swisssci crystal plate, using the Hydra liquid handler.

      2. Pipette the protein solution into a column in a V well microplate well plate.

      3. Set up a sitting drop plate comprising 288 drops, using the robotic liquid handler (Mosquito) using three protein:reservoir ratios (100 nL protein + 100 nL reservoir; 200 nL protein + 100 nL reservoir; 100 nL protein + 200 nL reservoir) (see Note 8).

      4. Seal completed plate using VIEWseal.

      5. Image and store plates by Fibonacci schedule in Formulatrix imager at 20°C (see Figure 4 for examples of crystals).

      6. Cut out a square of seal over the selected crystal drop, and open drop.

      7. Add 1 μL of cryoprotectant mix (30% glycerol or Peg 400 in original crystallization condition) onto crystals/drop. Table 1 gives examples of crystallized VHH-RBD complexes.

      8. Place mounting pin onto magnetic wand, loop crystal from drop, and place rapidly into foam dewar containing liquid nitrogen.

      9. Store mounted crystal in puck within foam dewar.

      10. Transfer puck into liquid nitrogen pre-cooled Dry shipper.


        Table 1. Examples of crystallized VHH-RBD complexes with details of crystallization conditions, the space group, and resolution limit of crystals generated.

        Complex RBD-F2 RBD-H3-C1 RBD-C5
        Crystal screen PactTM JCSGTM SG1TM
        Crystallisation condition 0.1 M SPG, pH 8, 25% Peg 1500 1.0 M Lithium chloride, 0.1 M Citrate pH 4, 20% Peg 6000 0.2 M Na Acetate, 0.1 M Na Cacodylate pH 6.5 and 30% w/v PEG 8000
        Time for crystal formation 3 days less than 24 h 3 days optimal growth
        Ratio 0.2 μL protein with 0.1 μL reservoir 0.2 μL protein with 0.1 μL reservoir 0.1 μL protein with 0.1 μL reservoir
        Concentration 34 mg/mL 18 mg/mL 18 mg/mL
        Cryoprotectant glycerol Peg400 glycerol
        Data collection
        Exposure 0.006s 0.012s 0.008s
        Space group P31 P41212 P21212
        Resolution (Å) 2.3 1.9 1.5



    Figure 4. Crystal shape of the different complexes: RBD-F2 (left), RBD-H3-C1 (middle), and RBD-C5 (right).

Notes

  1. Good quality DNA with minimum protein and chemical contamination should have ratios of absorbance 260/280 between 1.8–2.0, and 260/230 between 2.0–2.2.

  2. Our protocol is suitable for any scale of expression: 1–3 mL plate experiments, and 30–300 mL up-scaled expression in flasks. Scale provided volumes and quantities of reagents proportionally to the volume of transfected cells.

  3. Do not mix DNA and PEI directly, as they will precipitate immediately.

  4. The use of valproic acid, sodium propionate (histone deactylase inhibitors), and glucose feed in combination helps substantially enhance gene expression.

  5. It is important to check periodically that there is no precipitation of the VHH during the concentration step and to stop further concentrating the protein if this is observed. Any precipitate can be removed by centrifugation of the sample in a microfuge at 12,000 × g for 10 min and then measuring the final protein concentration by absorbance at 280 nm.

  6. A molar excess of VHH is added, to ensure that the RBD is fully complexed with the nanobody.

  7. EndoH has maximum efficiency at pH ~5.2. However, as the majority of proteins are unstable at this pH, the reaction is carried out at pH 7.5. Also, many proteins are unstable at 37°C for long periods of time, so RT is preferred for the reaction. EndoH is active at RT and at pH 7.5, but the reaction takes longer than it would if it was run under optimal conditions.

  8. The two sparse matrix screens (JCSG+ and SG1) were routinely screened at two different concentrations (usually, 34 mg/mL, and 18 mg/mL) with three different ratios. For the complexes RBD-F2 and RBD-H3-C1, we obtained a number of crystallization hits, while for RBD-C5 we only obtained one hit. Only occasionally the PACT screen was used. Crystals were harvested straight from the sparse matrix screen and did not require further optimization. To obtain more crystals with very subtle different diffraction quality, usually the same condition was set up as a row on the mosquito, using the same protocol. This also allowed testing of two different cryo-conditions (usually, glycerol, and Peg 400). Crystals were very reproducible. The cleanest crystals were harvested from all hits usually within 24 h of growth, but diffraction quality remained stable up to 5 days thereafter for most hits.

Recipes

  1. Valproic acid

    500 mg in 10 mL of cell media or HBSS, filter 0.2 μm, and store at -20°C.

  2. Propionate solution

    1 g sodium propionate in 10 mL of cell media or HBSS, filter, and store at -20°C.

  3. PEI

    Suspend 100 mg of PEI Max 40K in 90 mL of MilliQ water. Stir using a PTFE coated stirring bar. It should take less than 5 min to dissolve, then adjust the pH using NaOH or HCl to 7.0. Add water to 100 mL, filter 0.2 μm, and store at -20°C.

  4. Glucose

    45 g in 100 mL of HBSS or cell medium, filter 0.2 μm, and store at -20°C.

  5. Wash buffer

    PBS + 30 mM imidazole pH 7.4

  6. Elution buffer

    PBS + 300 mM imidazole pH 7.4

  7. Gel filtration buffers

    20 mM Tris, and pH 7.5, 200 mM NaCl, or 50 mM Tris pH 7, 150 mM NaCl

  8. TES buffer

    200 mM Tris pH 8, 0.5 mM EDTA, 500 mM sucrose

  9. TES/4 buffer

    50 M Tris pH 8, 125 mM sucrose

  10. TB medium

    Yeast extract (24 g/L), Tryptone (20 g/L), Glycerol (4 mL/L), Phosphate buffer (100 mL/L) of 0.17 M KH2PO4, 0.72 M K2HPO4

Acknowledgments

This work was supported by the Rosalind Franklin Institute, funding delivery partner EPSRC. and the Rosalind Franklin Institute EPSRC grant no. EP/ S025243/1. J.H.N., A.L.B. are supported by Wellcome Trust (100209/Z/12/Z). J.H. is supported by the EPA Cephalosporin and Edward Penley Abraham Funds. X-ray data were obtained using Diamond Light Source COVID-19 Rapid Access time on Beamline I03, I04 and I24 (proposal MX27031).

Competing interests

The Rosalind Franklin Institute has filed a patent on the identification of nanobodies to the spike protein of SARS-CoV-2; R.J.O., J.H. and J.H.N. are named as inventors. The other authors declare no competing interest.

References

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简介

摘要:SARS-CoV-2 刺突蛋白的受体结合域 (RBD) 与上皮细胞表面的血管紧张素转化酶 2 (ACE-2) 结合,导致病毒融合并进入细胞。 这种相互作用可以通过美洲驼衍生的纳米抗体 (VHH) 与 RBD 的结合来阻止,从而导致病毒中和。 通过 X 射线晶体学对 VHH-RBD 复合物进行结构分析,可以精确定位 VHH 表位,并解释和预测变异突变的影响。 对此的关键是 VHH-RBD 复合物的可重复生产和结晶的协议。 根据我们的经验,我们描述了表达和纯化蛋白质的工作流程,以及产生 VHH-RBD 复合物衍射质量晶体的筛选条件。 蛋白质复合物的生产和结晶大约需要十二天,从构建载体到收获和冷冻晶体以收集数据。


背景

骆驼(美洲驼、羊驼和骆驼)产生一种独特类型的仅重链抗体,该抗体包含与 Fc 恒定结构域(CH2 和 CH3)连接的可变结构域 (VHH)。 VHH 可以作为单域抗原结合蛋白或纳米抗体生产( Muyldermans,2013 年) ,在生命科学中有广泛的应用(Pleiner等人,2015 年;Jovcevska 和 Muyldermans,2020 年) 。为应对 COVID-19 大流行(Dhama等人,2020),我们和许多其他小组开发了 VHH 试剂,可与 SARS-CoV-2 刺突蛋白的受体结合域 (RBD) 结合,从而阻断与 ACE 的结合-2 在细胞表面,这是导致细胞感染的主要相互作用(Xiang等人,2020;Hanke等人,2020;Schoof等人,2020;Koenig等人,2021;Huo等人。 ,2020 年,2021 年) 。这些 VHH 的多聚体版本,无论是三聚体还是 IgG Fc 融合体,已被证明可以在体外和 COVID-19 动物模型中中和 SARS-CoV-2 (Huo等人,2021;Nambulli等人,2021) . VHH-RBD 复合物的结构分析揭示了表位聚集的两个区域,一个位于或接近 ACE-2 结合表面(簇 2),一个位于 RBD 的另一侧(簇 1) (Tang等人,2014 年)。 , 2021) 。关于 VHH 结合位点的信息能够解释和预测刺突蛋白突变的影响。根据我们的经验,一些 VHH-RBD 复合物的结晶需要与两个与正交位点结合的 VHH 形成 RBD 复合物(Huo等人,2021) 。
VHH 通常在大肠杆菌菌株 WK6 中作为六组氨酸标签蛋白产生,使用异丙基 β-D-1-硫代半乳糖吡喃糖苷 ( ITPG) 诱导型启动子,并添加分泌信号(例如,pelB 或 ompA),以实现恢复诱导表达后来自周质的VHH。初级纯化使用固定化金属亲和层析 (IMAC),并添加凝胶过滤作为精制步骤。或者,可以直接从裂解细胞中纯化 VHH,但根据我们的经验,通过渗透休克提取周质可提供更高的蛋白质产量。
作为一种糖基化蛋白,SARS-CoV-2 刺突蛋白 RBD 的产生需要在昆虫和哺乳动物细胞都已使用的高等真核细胞中表达。将适当的信号序列添加到 N 末端以将产物引导至细胞培养基,并将六组氨酸标签添加到 C 末端以通过 IMAC 纯化。 RBD(Asn331 和 Asn343)的 N-糖基化引入了化学异质性,特别是如果在哺乳动物细胞中产生,这通常会抑制结晶( Chang等人,2007)。通过在存在甘露糖苷酶抑制剂 kifunensine 的情况下培养细胞,可以将 N-糖基化抑制在高甘露糖状态(GlcNAc2Man9 糖型)。随后可以通过用内切糖苷酶 F1 或 H 处理将这些残基修剪回单个 N-乙酰氨基葡糖残基(Chang et al. , 2007) 。根据过去的经验(Nettleship et al. , 2013),这是在纯化 VHH-RBD 复合物之前最有效地进行的。
通过 IMAC 从细胞培养基中纯化分泌的糖蛋白的一个问题是,一些培养基成分在层析步骤中置换了带 His 标签的目标蛋白,从而显着降低了产量。在这里,我们使用了一种通过 IMAC 和凝胶过滤进行亲和纯化的自动化方法,该方法包括将样品分批加载到 Ni-NTA 柱上,每批之间都有一个柱清洗步骤(Nettleship等人,2009) 。
我们已经报告了使用市售屏幕、96 孔格式和 1-200 nL 的低样品体积的许多 VHH-RBD 复合物的结晶。使用同步加速器 X 射线从初级晶体命中收集衍射数据,并通过分子置换解决结构 (Huo et al. , 2020 , 2021 )。获得优质晶体的关键是蛋白质的制备。因此,在本文中,我们描述了用于生产 VHH-RBD 配合物及其结晶的优化工作流程,以便通过 X 射线晶体学进行分析。

关键字:SARS-CoV-2, 纳米抗体, 受体结合域, 蛋白质纯化, 结晶

材料和试剂
受体结合结构域的产生
向量构造
用 KpnI/PmeI 消化的 pOPINTTG 载体(图 1A)
人类密码子优化的合成 RBD 基因(编码氨基酸 330 – 352)具有 15 bp 延伸与 pOPINTTG 融合进入位点重叠(小写):
5'gcgtagctgaaaccggcCCGAATATCACAAATCTTTGTCCTTTCGGAGAAGTATTCAACGCAACTCGCTTCGCATCAGTATATGCCTGGAACCGCAAACGAATTTCTAATTGTGTCGCCGACTACTCTGTGCTTTACAACTCAGCATCATTTTCAACATTCAAATGCTATGGTGTCTCCCCTACGAAGTTGAACGATCTTTGTTTCACTAATGTCTACGCCGATTCCTTTGTTATTAGGGGGGACGAGGTTCGCCAGATCGCGCCGGGCCAAACGGGCAAGATAGCTGACTATAATTATAAGCTGCCAGACGACTTTACAGGCTGCGTTATCGCTTGGAATTCAAACAATCTTGATAGCAAAGTGGGTGGTAATTATAACTACCTCTACAGACTGTTTCGCAAGTCTAATCTGAAGCCTTTCGAGCGGGACATCTCTACTGAGATCTATCAAGCTGGTTCAACCCCCTGCAATGGCGTCGAGGGTTTTAACTGTTACTTCCCACTTCAGTCATACGGATTTCAACCAACTAATGGGGTTGGCTATCAGCCGTACCGCGTGGTCGTTCTTAGCTTTGAGCTGCTTCACGCCCCTGCAACGGTGTGCGGACCGAAAAAAAGTACAAAaaacatcaccatcac 3'
ClonExpress II One Step Cloning 试剂盒(Vazyme,目录号:C113-02)
NucleoSpin 148 ® Gel and PCR Clean-up kit(MACHEREY-NAGEL,目录号:12303368)
恒星感受态细胞(Takara Bio,目录号:636766)
LB培养基(Sigma-Aldrich,目录号:L3022)
SOC(具有分解代谢物抑制的超级最佳肉汤)恢复培养基(ThermoFisher Scientific,目录号:15544034)
氨苄青霉素(Sigma-Aldrich,目录号:A9393)
LB-琼脂板添加 100 μg /mL 氨苄青霉素
质粒小量制备试剂盒(例如,Qiagen,目录号:27104)
Plasmid Plus Midi 试剂盒(例如,Qiagen,目录号:12941)
测序引物:pTTfwd 5' TCCACAGGTGTCCACTCC 3'
pTTrev 5' TCCTTTATTAGCCAGAGG 3'


expi293 TM细胞中的表达
500 mL 带通风封口的无菌挡板烧瓶(ThermoFisher Scientific,目录号:4116-0500)
Countess TM细胞计数室载玻片(ThermoFisher Scientific,目录号:C10228)
Expi293 TM电池(ThermoFisher Scientific,目录号:A14527)
Expi293 TM表达培养基(ThermoFisher Scientific,目录号:A1435101)
台盼蓝溶液(ThermoFisher Scientific,目录号:15250061)
汉克斯平衡盐溶液(HBSS) (10 × )(ThermoFisher Scientific,目录号:14185052)
Gibco OPTI-MEM 减少血清培养基(ThermoFisher Scientific,目录号:31985062)
丙戊酸(Sigma-Aldrich,目录号:P4543)
葡萄糖(45%溶液)(参见配方或来自Sigma-Aldrich,目录号:G8769)
丙酸钠(Sigma-Aldrich,目录号:P1880)
PEI MAX 40 K(Polysciences Inc.,目录号:24,765-1)
Kifunensine(Sigma-Aldrich,目录号:K1140)
预制 SDS 聚丙烯酰胺凝胶(例如,NuPAGE TM 10%,Bis-Tris ThermoFisher Scientific ,目录号 WG1201A)
InstantBlue ®考马斯蛋白染色剂(Abcam,目录号:ab119211)
RBD纯化
5 ml HisTrap_FF Ni-NTA 柱(Cytiva,目录号:1752860)
SD75 16/600 尺寸排除柱(Cytiva,目录号:28989333)
咪唑(Sigma-Aldrich,目录号:I2399)
PBS,磷酸盐缓冲盐水,10 ×溶液(Fisher,目录号:BP399-20)
洗涤缓冲液(见配方)
洗脱缓冲液(参见配方)
凝胶过滤缓冲液(参见配方)


VHH 生产
向量构造
用 SfiI 切割的 pADL-23c 载体(图 1B)(New England Biolabs,目录号: R0123S)
输液引物:
VHH Fwd 引物 5' GTTATTACTCGCGGCCCAGCCGGCCATGGCCC 3'
VHH Rev 引物 5' GGTGATGGTGTTGGCCTTTATTAATGATGGTGGTGATGGTG 3'
校对聚合酶(例如,PhusionFlash TM高保真聚合酶 ThermoFisher Scientific,目录号:F548S)
pADL-23c 测序引物:PhdseqFwd 5' GCTTCCGGCTCGTATGTTG 3'
PhDseqRev 5' GTCGTCTTTCCAGACGTTAG 3'
2 mL 冷冻管(例如ThermoFisher Scientific,目录号:5000-0020 )
大肠杆菌表达
WK6 细胞大肠杆菌(Migula)Castellani 和 Chalmers(ATCC,目录号:47078)
极好的肉汤(TB)培养基(见食谱)
异丙基β-D-1-硫代吡喃半乳糖苷,IPTG(Sigma-Aldrich,目录号:I6758)
葡萄糖
氯化镁
氨苄青霉素
VHH 纯化
TES 缓冲液(见配方)
DNase I(Sigma-Aldrich,目录号: D4263)
氯化镁(Sigma-Aldrich,目录号: M8266)
蔗糖(Sigma-Aldrich,目录号:S0389)




 


图 1. 矢量图 (A) pOPINTTGneo (B) pADL-23c


VHH-RBD 复合物的结晶


VHH-RBD复合物的制备
EndoH(New England Biolabs,目录号:P0702S)
SD200 10/300 尺寸排除柱(Cytiva,目录号:28990944)
结晶筛选
Swisssci Triple-Drop 结晶板(Molecular Dimensions,目录号: MD11-003-100)
无盖 V 孔微孔板,天然,聚丙烯(Greiner,目录号:651201 96)
VIEWseal TM板密封剂,透明,不可刺穿的 Greiner,目录号:676070)
Pact Premier TM结晶筛选条件(Molecular Dimension,目录号: MDSR-29)
JCSG-plus TM结晶筛选条件(Molecular Dimensions,目录号:MDSR-37)
SG1 TM结晶条件(Molecular Dimensions,目录号: MDSR-88 )
晶体的冷冻保存
甘油(分子尺寸,目录号: MD2-100-65 )
PEG 400(Sigma-Aldrich,目录号:06855)
安装的圆形LithoLoops(0.25 mm) (分子尺寸,目录号: MD7-137 )
安装的圆形 LithoLoops(0.15 毫米)(分子尺寸目录号:MD7-135)
标准泡沫杜瓦瓶(分子尺寸,目录号:MD7-35)
Uni-Puck 10 Pack(分子尺寸,目录号:MD7-613)
Cryotool 套件(Molecular Dimensions,目录号:MD7-517)
Dry Shipper(分子尺寸,目录号:MD7-21)

设备
微量分光光度计(例如,ThermoFisher Scientific、NanoDrop TM One/OneC 微量紫外-可见分光光度计:ND-ONE-W)
CO 2轨道摇床(例如,n-Biotek.com ANICELL 培养箱,目录号:NB-206CXL/NB)
适用于 50 mL Falcon 管的台式离心机(Sorvall Legend RT Plus)
细胞计数器(例如,ThermoFisher Scientific Countess TM 3 自动细胞计数器)
ÄKTA Xpress 自动化多步纯化系统
液体处理器(例如,Art Robbins Instruments 的 Hydra Dispenser)
用于结晶板的小容量分配器(例如,SPT labtech 蚊子® LV)
晶体板成像仪(例如,Formulatrix RockImager ®系统)




程序


受体结合结构域的产生


向量构造
对于 10 μL的融合反应,混合 20 ng (1-3 μL) 合成基因、100 ng (1-2 μL) PmeI/NcoI cut pOPINTTG 载体、1 μL Exnase II和 2克隆试剂盒随附的 μL 优化缓冲液。
将反应在 37 °C 下孵育30 分钟,然后立即停止,加入 20 μL冰冷的 TE 缓冲液。
使用 5 μL所得反应混合物转化 20 μL Stellar 感受态细胞,在冰上孵育 30 分钟,然后在 42 °C 下热休克45 秒。
加入 400 μL的 SOC 培养基并在 37 °C下孵育45 分钟。
100 μ L 的细胞和培养基混合物放在 LB 琼脂板上,辅以 100 μ g/mL 氨苄青霉素,并在 37 °C 下培养过夜。
将单个菌落挑入含有 100 μg /mL 氨苄青霉素的 3 mL LB 中,并在 37 °C下孵育过夜。
通过将 0.5 mL 的 50% (v/v) 无菌甘油添加到 2 mL 冷冻管中 LB 中细胞的 0.5 mL 过夜培养物中,并在 -80 °C 下冷冻,生产甘油库存。
从沉淀的细胞中制备 DNA,并通过使用 pTTfwd 和 PTTrev 引物对小量制备 DNA 进行测序来确认正确的克隆。
使用 50% 甘油原液培养更大规模的培养物,用于转染级质粒制备。
转染级质粒(0.5-1 mg ),如果需要,将质粒储存在 -20 °C的无菌 1.5 mL Eppendorf 管中。
使用 NanoDrop 分光光度计测量 DNA 的纯度和浓度。用于转染的质粒 DNA 应该是高纯度的(见注 1)。
DNA 的浓度和纯度由仪器使用以下等式自动计算:
浓度:DNA (µg/mL) = (A 260读数 – A 320读数) × 稀释倍数 × 50 µg/mL
纯度:(A 260 /A 280 )=(A 260读数 - A 320读数)÷(A 280读数 - A 320读数)
每百万个转染细胞使用 1 μ g DNA。
转染方案
Expi293 TM细胞在加湿 (80%) 培养箱中解冻后保持悬浮培养至少 3 代(3-30 代可用于实验),5-8% CO 2 ,37 °C ,开在 Gibco Expi293 TM Express 培养基中以 120 rpm 的转速摇床,细胞密度在 0.5 和 5.0 × 10 6 个细胞/mL 之间。使用 125 mL 烧瓶来维持 30 mL 的 Expi293 细胞。
转染前一天,以 1 × 10 6 个细胞/mL的细胞密度接种 Expi293 TM细胞。对于 RBD 的每次制备,在 500 mL 烧瓶中设置三个 170 mL 的培养物。
在转染当天,确定细胞计数和存活率,如果细胞计数在 2 × 10 6 –2.5 × 10 6 /mL 之间且存活率至少为 95%,则继续转染(见注 2)。
对于每个 170 mL 培养物,将 17 mL OPTI-MEM 培养基与 170 μg质粒 DNA 和 918 μL PEI Max 40kDa 转染试剂混合在 50 mL Falcon 管中。
彻底混合,在室温 (RT) 下孵育 10 分钟,然后轻轻(逐滴)加入 Expi293 TM细胞(见注 3)。
从 1 mg/mL 的原液浓度(每 100 mL 培养体积 100 μL)中添加 kifunensine ,并在125 rpm、5 – 8% CO 2 、80% 湿度和 37 °下将细胞返回到轨道振荡器上孵育C. _
16-18 小时后,在每 170 mL培养物中加入 2,890 μL丙戊酸、1,100 μL丙酸钠和 3,400 μL葡萄糖。将培养物送回培养箱(见注 4)。
在转染后第 5 天,确定细胞计数和活力。
收获培养基,在 0.5-L 离心瓶中以 6,000 × g离心20 分钟。使用 0.45 μ m 0.5-L 瓶顶过滤器进行过滤消毒。
纯化
IMAC-SEC 纯化方案适用于 ÄKTA Xpress 平台,使用 Nettleship等人中描述的程序。 (2009 年)。相同的工作流程可以在其他具有自动峰检测的纯化系统上实施。独角兽计划的成绩单作为附录提供。
使用“凝胶过滤平衡”程序和凝胶过滤缓冲液(20 mM Tris,pH 7.5,200 mM NaCl)平衡凝胶过滤柱。
按照“哺乳动物准备系统”的方法设置系统。它将泵清洗 A1 和 A2,并清洁入口。然后它会要求您将 5-mL 色谱柱拧入位置 1,并平衡色谱柱。
在过滤后的细胞上清液中加入等体积的 PBS,用 NaOH 调节 pH 至 7.4。
插入色谱柱并准备好上样后,从缓冲液中缓慢取出 A2 管线(通过小幅度移动以避免管线中出现气泡),然后将其插入样品中。确保所有的线条都在瓶子的底部。
让协议在一夜之间运行。
使用来自 ÄKTA 的迹线,选择包含目标蛋白质的正确级分。将 10 μL 的蛋白质溶液与 10 μL 的样品缓冲液混合,并在 95 °C 下加热5 分钟。运行样品的 SDS-PAGE 凝胶,并使用 Instant Blue ®对其进行染色。
使用 10 kDa 浓缩离心机在 2,500 × g和 4 °C 下将蛋白质浓缩至 5 mg/mL 。
分装(例如,0.1 mL)并通过将试管插入液氮中快速冷冻蛋白质。
储存在 -80 °C 。


生产 VHH


向量构造
通过筛选 M13 噬菌体展示文库鉴定的 VHH 被重新表达用于蛋白质生产。
VHH Fwd 引物和 VHH Rev 引物以及以下 PCR 条件从源展示载体中扩增 VHH:
1) 98°C 10 秒
2) 30 个循环:
98°C 1 秒
60°C 5 秒
72°C 15 秒
3) 72°C 2 分钟
4) 4°C 保持
根据制造商的说明使用 Nucleospin ®试剂盒进行提取。如克隆 RBD 所述,将PCR 产物克隆到SfiI 切割的 pADL-23c 载体中。
通过使用 PhDseqFwd 和 PhDseqRev 引物测序来验证克隆。
在大肠杆菌中的表达 
如上所述转化化学感受态 WK6 细胞,用于构建 RBD 表达载体。
从转化细胞板中挑选一个菌落,并设置预培养:8 mL 的 TB 辅以 100 μg/mL 氨苄青霉素、2% 葡萄糖和 1 mM MgCl 2。
设置培养:在 2-L 烧瓶中加入 800 mL 的 TB 培养基,辅以 100 μg/mL 氨苄青霉素、0.1% 葡萄糖和 1 mM MgCl 2。
将烧瓶/培养基预热至 37 °C 。
°C的轨道摇床上添加预培养和培养培养。
600达到 ±1.2(通常需要大约 3.5 小时)时,将 IPTG 添加到 1 mM 的最终浓度,并在 225 rpm 和 28 °C下继续生长过夜。
在 4 °C 下以 2,500 × g将细胞沉淀 15分钟。
VHH 的纯化
将 15 mL 的 TES 缓冲液添加到细胞颗粒中,并在 4 °C 下用磁力搅拌器缓慢重新悬浮在瓶子中过夜。
第二天,在重悬培养物中加入两倍体积的冷TES/4 缓冲液,辅以 120U Kunitz DNaseI,缓慢搅拌 2 小时。
使用 TES/4 缓冲液最多加注 80 mL。
以 28,000 × g离心沉淀 和 4 °C 30 分钟(在 50 mL 管中)。
在真空下通过 0.8 μm 过滤器过滤上清液。
用五倍体积的 PBS (pH 7.4) 稀释上清液,并充分混合。
将样品加载到两个串联的 5 mL IMAC 柱上,速度 <2 mL/min。
用含有 30 mM 咪唑的 PBS (pH 7.4) 清洗柱子。
用含有 300 mM 咪唑的 PBS 以 1 mL/min 的速度将样品洗脱到 96 孔收集板中,收集 1 mL 分数。
池 A 280个峰值分数(约 7.5 mL)并在凝胶过滤缓冲液(50 mM Tris pH 7、150 mM NaCl)中的 Superdex S75 16/600上运行。
汇集 A 280个峰值蛋白质组分,并使用 5 kDa MWCO 浓缩器浓缩,通常浓缩至 15 mg/mL(见注 5)。 
分装(例如,0.1 mL)并通过将试管插入液氮中快速冷冻蛋白质。
储存在 -80 °C 。


VHH-RBD 复合物的结晶


VHH:RBD 复合物的制备
将 5 mg RBD (5 mg/mL) 与 3 mg VHH (15 mg/mL) 以 RBD:VHH 为 1:1.2 的摩尔比混合,并在冷藏室中以 2 rpm 搅拌孵育 3 小时(见注 6)。
将 RBD-VHH 复合物与 0.4 mg EndoH 糖苷酶 (1 mg/mL) 在室温下以 2 rpm 搅拌过夜(见注 7)。
使用 5 kDa MWCO 浓缩器将混合物浓缩至 1 mL,并注入凝胶过滤缓冲液(50 mM Tris pH 7、150 mM NaCl)中的 Superdex 200 10/300。
器监测 A 280 、汇集峰级分并浓缩至 20 mg/mL(参见图 2 和 3)。


 


SD200 20/300 尺寸排阻柱(CV 23.56 mL)上纯化 VHH-RBD 复合物。 




 


图3. RBD(泳道 1)、VHH(泳道 2)和 RBD-VHH 复合物凝胶过滤部分的SDS页,用糖苷内切酶 HH 处理后(泳道 3-12) 。


结晶筛选
25 μ L 的结晶溶液从 96 孔母块中分配到 Swisssci 晶体板的储液孔中。
将蛋白质溶液移液到 V 井微孔板中的一列中。
设置一个包含 288 滴的坐式滴板,使用机器人液体处理器 (Mosquito),使用三种蛋白质:水库比率(100 nL 蛋白质 + 100 nL 水库;200 nL 蛋白质 + 100 nL 水库;100 nL 蛋白质 + 200 nL 水库)(见注 8)。
使用 VIEWseal 密封完成的板。
° C下,在 Formulatrix 成像仪中按斐波那契计划对板进行成像和存储(请参见图 4 中的晶体示例)。
在选定的水晶滴上切出一个正方形的密封,然后打开滴。
将 1 μ L 冷冻保护剂混合物(原始结晶条件下的 30% 甘油或 Peg 400)添加到晶体/滴上。表 1 给出了结晶 VHH-RBD 复合物的例子。
将安装销放在磁棒上,从水滴中循环晶体,然后迅速放入含有液氮的泡沫杜瓦瓶中。
将安装的水晶存放在泡沫杜瓦瓶内的冰球中。
将冰球转移到液氮预冷的干式运输机中。


表 1. 结晶 VHH-RBD 配合物的示例,详细说明了结晶条件、空间群和生成的晶体的分辨率限制。


复杂的 RBD-F2 RBD-H3-C1 RBD-C5
水晶屏风 契约TM JCSG TM SG1 TM 
结晶条件 0.1 M SPG,pH 8,25% Peg 1500 1.0 M 氯化锂、0.1 M 柠檬酸盐 pH 4、20% Peg 6000 0.2 M 醋酸钠、0.1 M 二甲胂酸钠 pH 6.5 和 30% w/v PEG 8000
晶体形成时间 3天 少于 24 小时 3天最佳生长
比率 0.2 μ L 蛋白质和 0.1 μ L 储液罐 0.2 μ L 蛋白质和 0.1 μ L 储液罐 0.1 μ L 蛋白质和 0.1 μ L 水库
浓度 34 毫克/毫升 18 毫克/毫升 18 毫克/毫升
冷冻保护剂 甘油 PEG400 甘油
数据采集  
接触 0.006s 0.012s 0.008s
空间群 P31 P41212 P21212
分辨率 ( Å) 2.3 1.9 1.5




 


图 4. 不同配合物的晶体形状:RBD-F2(左)、RBD-H3-C1(中)和 RBD-C5(右)。
笔记


具有最少蛋白质和化学污染的优质 DNA 的吸光度比应为 260/280 介于 1.8 – 2.0 之间,以及 260/230 介于 2.0–2.2 之间。
我们的方案适用于任何规模的表达:1 – 3 mL 板实验和 30 – 300 mL 在烧瓶中的放大表达。 Scale 提供与转染细胞体积成比例的试剂体积和数量。
不要直接混合 DNA 和 PEI,因为它们会立即沉淀。
丙戊酸、丙酸钠(组蛋白脱乙酰酶抑制剂)和葡萄糖饲料的组合使用有助于显着增强基因表达。
重要的是要定期检查在浓缩步骤期间没有 VHH 沉淀,如果观察到这种情况,则停止进一步浓缩蛋白质。任何沉淀物都可以通过在微量离心机中以 12,000 × g离心10 分钟来去除。然后通过 280 nm 处的吸光度测量最终蛋白质浓度。
添加摩尔过量的 VHH,以确保 RBD 与纳米抗体完全复合。
EndoH 在 pH ~5.2 时具有最高效率。然而,由于大多数蛋白质在此 pH 值下不稳定,因此反应在 pH 值 7.5 下进行。此外,许多蛋白质在 37°C 下长时间不稳定,因此反应首选 RT。 EndoH 在 RT 和 pH 7.5 时具有活性,但反应时间比在最佳条件下运行时要长。
两种稀疏矩阵筛选(JCSG+ 和 SG1)通常以两种不同的浓度(通常为 34 mg/mL 和 18 mg/mL)以三种不同的比率进行筛选。对于配合物 RBD-F2 和 RBD-H3-C1,我们获得了许多结晶命中,而对于 RBD-C5,我们只获得了一个命中。只是偶尔使用 PACT 屏幕。晶体直接从稀疏矩阵屏幕中收获,不需要进一步优化。为了获得更多具有非常细微不同衍射质量的晶体,通常使用相同的协议将相同的条件设置为蚊子上的一排。这也允许测试两种不同的低温条件(通常是甘油和 Peg 400)。晶体的重现性非常好。通常在生长后 24 小时内从所有命中中收获最干净的晶体,但对于大多数命中而言,衍射质量在此后 5 天内保持稳定。




食谱


丙戊酸
500 mg 在 10 mL 细胞培养基或 HBSS 中,过滤 0.2 μ m,并储存在 - 20 °C。


丙酸盐溶液
在 10 mL 细胞培养基或 HBSS 中加入 1 g 丙酸钠,过滤并储存在-20 °C。



将 100 毫克 PEI Max 40K 悬浮在 90 毫升 MilliQ 水中。使用 PTFE 涂层搅拌棒搅拌。溶解时间应少于 5 分钟,然后使用 NaOH 或 HCl 将 pH 值调节至 7.0。加水至 100 mL,过滤 0.2 μm , -20 °C保存。


葡萄糖
45 g 于 100 mL HBSS 或细胞培养基中,过滤 0.2 μm , -20 °C储存。


洗涤缓冲液
PBS + 30 mM 咪唑 pH 7.4


洗脱缓冲液
PBS + 300 mM 咪唑 pH 7.4


凝胶过滤缓冲液
20 mM Tris 和 pH 7.5、200 mM NaCl 或50 mM Tris pH 7、150 mM NaCl


TES 缓冲液
200 mM Tris pH 8、0.5 mM EDTA、500 mM 蔗糖


TES/4 缓冲器
50 M Tris pH 8、125 mM 蔗糖


结核培养基
酵母提取物 (24 g/L)、胰蛋白胨 (20 g/L)、甘油 (4 mL/L)、磷酸盐缓冲液 (100 mL/L) 的 0.17 M KH 2 PO 4 、0.72 MK 2 HPO 4 




致谢


这项工作得到了资助交付合作伙伴 EPSRC 罗莎琳德富兰克林研究所的支持。和罗莎琳德富兰克林研究所 EPSRC 授权号。 EP/S025243/1。 JHN、ALB 由 Wellcome Trust (100209/Z/12/Z) 提供支持。 JH 得到 EPA 头孢菌素和 Edward Penley Abraham 基金的支持。 X 射线数据是在光束线 I03、I04 和 I24 上使用钻石光源 COVID-19 快速访问时间获得的(提案 MX27031)。




利益争夺


罗莎琳德·富兰克林研究所已申请鉴定 SARS-CoV-2 刺突蛋白纳米抗体的专利; RJO、JH 和 JHN 被命名为发明人。其他作者声明没有竞争利益。




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引用:Le Bas, A., Mikolajek, H., Huo, J., Norman, C., Dormon, J., Naismith, J. H. and Owens, R. J. (2022). Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein. Bio-protocol 12(9): e4406. DOI: 10.21769/BioProtoc.4406.
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