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Production of the Receptor-binding Domain of the Viral Spike Proteins from 2003 and 2019 SARS CoVs and the Four Common Human Coronaviruses for Serologic Assays and Inhibitor Screening

从2003年和2019年SARS冠状病毒和四种常见人类冠状病毒的血清学分析和抑制剂筛选中产生病毒刺突蛋白受体结合域

BS Bruno Segovia-Chumbez
SG Stephen D. Graham
RJ Ramesh Jadi
AS Aravinda M. de Silva
LP Lakshmanane Premkumar

发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4026 浏览次数: 6747

评审: Luis Alberto Sánchez VargasSesha Lakshmi Arathi PaluriVikas Duhan

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Jun 2020

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Abstract

The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification of properly folded RBD proteins from SARS and common-cold HCoVs in mammalian cells. RBD proteins were produced with cleavable tags for affinity purification from the cell culture medium and to support multiple immunoassay platforms and drug discovery efforts.


Graphic abstract:



High-Yield Production of Viral Spike RBDs for Diagnostics and Drug Discovery


Keywords: SARS-CoV-2 (SARS-CoV-2) search SARS-CoV (SARS-CoV) search Coronavirus (冠状病毒) search COVID-19 (2019冠状病毒病) search Antigen (抗原) search Immunoassay (免疫测定) search Halo-tag (Halo-tag) search Spike protein (纤突蛋白) search Antibody (抗体) search Inhibitor screening (抑制剂的筛选) search Drug discovery (药物研发) search

Background

The receptor-binding domain (RBD) of the coronavirus spike protein is critical for viral attachment, fusion, and entry. It is also the primary target for antibody response and the development of entry inhibitors and vaccines. The RBDs of 2003 and 2019 SARS CoVs and the four common endemic human CoVs are poorly conserved, representing a promising antigen for detecting viral-specific antibodies in humans. We have recently shown that the RBD of SARS-CoV-2 is highly sensitive and specific for detecting antibodies nine days after the onset of symptoms (Premkumar et al., 2020). Levels of RBD-binding antibodies in human sera are strongly correlated with the SARS-CoV-2 neutralizing titer in patients. Thus, RBD-based serologic assays are attractive to identify individual and environmental risk factors for severe illness and to monitor SARS-CoV-2 transmission in the community. Prior immunity to common human endemic coronaviruses (229E, NL63, OC43, and HKU1) has been reported to enhance the inflammatory response to SARS-CoV-2 (Grifoni et al., 2020; Mateus et al., 2020). Here, we present a detailed step-by-step method for expressing and purifying the RBD of 2003 and 2019 SAR CoVs and the four common endemic human CoVs for serologic assays and inhibitor screening (Premkumar et al., 2020 and Puhl et al., 2021). The technique allows the production of RBDs fused to a TEV protease cleavable self-labeling protein (HaloTag) at the N-terminus and a Twin-Strep-tag and a His-tag at the C-terminus. The tags were designed to aid affinity purification and oriented capture of antigens on solid supports incorporating streptactin, streptavidin, or nickel-nitrilotriacetic acid. The current protocol utilizes a mammalian expression system (Expi293) to produce milligram quantities of recombinant RBDs from a small cell culture volume within 5-7 days, using a single affinity purification step.


Materials and Reagents

  1. Cryopreservation Tubes (Thermo Scientific, catalog number: 374081)

  2. Poly-Prep Chromatography Columns (Bio-Rad, catalog number: 7311550)

  3. Econo-Pac Chromatography Columns (Bio-Rad, catalog number: 7321010)

  4. SnakeSkin Dialysis Tubing (Thermo Fisher Scientific, catalog number: 68700)

  5. SnakeSkin Dialysis Clips (Thermo Fisher Scientific, catalog number: 68011)

  6. Staples 2” Binder Clips, Large (Staples, catalog number: 10669)

  7. 5 ml Serological pipette (Thermo Fisher Scientific, catalog number: 13-678-11D)

  8. 10 ml Serological pipettes (Thermo Fisher Scientific, catalog number: 13-678-11E)

  9. 50 ml Falcon tube (Cell star, catalog number: 22761)

  10. Precision Plus Protein Kaleidoscope 500 ml (Bio-Rad, catalog number: 161-0375)

  11. Any kD Mini-PROTEAN TGX Stain-Free Protein Gels, 12-well, 20 μl (Bio-Rad, catalog number: 4568125)

  12. 2× Laemmli Sample Buffer (Bio-Rad, catalog number: 1610737)

  13. Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 2-gel (Bio-Rad, catalog number: 1658005)

  14. 2-Mercaptoethanol, 10 ml (Sigma-Aldrich, catalog number: M6250)

  15. Coomassie Brilliant Blue R-250 Dye (Thermo Fisher Scientific, catalog number: 20278)

  16. Expi293 Expression System Kit (Thermo Fisher Scientific, catalog number: A14635)

    Note: Store cells in liquid nitrogen and other reagents at 2°C to 8°C.

  17. Mr. Frosty (Thermo Scientific, catalog number: 5100-0001)

  18. Ni-NTA Agarose (QIAGEN, catalog number: 30230). Store at 2°C to 8°C

  19. DMSO (Millipore Sigma, catalog number: 41640)

  20. Opti-MEM Medium (Thermo Fisher Scientific, catalog number: 11-058-021)

  21. Tris (MP, catalog number: 103133)

  22. NaCl (Fisher, catalog number: S271-10)

  23. Glycerol (VWR, catalog number: BHD 1172-1LP)

  24. Sucrose (Fisher, catalog number: BP-220-1)

  25. Imidazole (Thermo Fisher Scientific, catalog number: 03196-500)

  26. Liquid nitrogen (Arc gases)

  27. Purify Buffer (see Recipes)

  28. Elution Buffer (see Recipes)

  29. Dialysis Buffer (see Recipes)

  30. Stain Buffer for SDS PAGE (see Recipes)

  31. Destain Buffer for SDS PAGE (see Recipes)

Equipment

  1. Fisherbrand Shaker Flasks, Plain Bottom, Vented (Thermo Fisher Scientific, catalog number: PBV12-5). Store at room temperature

  2. 4 L Beaker (Thermo Fisher Scientific, catalog number: 02-555-25K)

  3. Forma Steri-Cycle i160 CO2 Incubator (Thermo Fisher Scientific, Forma, catalog number: 51030301)

  4. CO2 Resistant Shaker (Thermo Fisher Scientific, catalog number: 88881101)

  5. Biological safety cabinet (Labguard, Class II, Type A2)

  6. Precision Water Bath GP 15D–5 L and 10 L (Thermo Scientific, catalog number: TSGP15D)

  7. Magnetic stir bar (Thermo Fisher Scientific, catalog number: 14-512-136)

  8. Centrifuge (Sorvall, model: RC-5B)

  9. Centrifuge (Eppendorf, model: 5810 R)

  10. Centrifuge (Thermo, model: Sorvall Legend Micro 21R)

  11. Freezer (Thermo Scientific Revco RLE60086A -86 °C)

  12. Cryogenic dewar (Cole Parmer)

  13. Mini-PROTEAN® Tetra Vertical Electrophoresis Cell for Mini Precast Gels (Bio-Rad, Catalogue number: 1658004)

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Segovia-Chumbez, B., Graham, S. D., Jadi, R. S., de Silva, A. M. and Premkumar, L. (2021). Production of the Receptor-binding Domain of the Viral Spike Proteins from 2003 and 2019 SARS CoVs and the Four Common Human Coronaviruses for Serologic Assays and Inhibitor Screening. Bio-protocol 11(10): e4026. DOI: 10.21769/BioProtoc.4026.
  2. Premkumar, L., Segovia-Chumbez, B., Jadi, R., Martinez, D. R., Raut, R., Markmann, A., Cornaby, C., Bartelt, L., Weiss, S., Park, Y., Edwards, C. E., Weimer, E., Scherer, E. M., Rouphael, N., Edupuganti, S., Weiskopf, D., Tse, L. V., Hou, Y. J., Margolis, D., Sette, A., Collins, M. H., Schmitz, J., Baric, R. S. and de Silva, A. M. (2020). The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci Immunol 5(48): eabc8513.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 抗体分析 > 抗体检测

免疫学 > 抗体分析 > 抗体-抗原相互作用

生物化学 > 蛋白质 > 表达

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