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

Bruno  Segovia-Chumbez; Stephen D. Graham; Ramesh  Jadi; Aravinda M. de Silva; Lakshmanane Premkumar

doi:10.21769/BioProtoc.4026

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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 email

LP Lakshmanane Premkumar email

发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4026 浏览次数: 5739

评审: Luis Alberto Sánchez VargasSesha Lakshmi Arathi PaluriVikas Duhan

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The authors used this protocol in:

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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)

Inhibitor screening (抑制剂的筛选)

Drug discovery (药物研发)

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

Cryopreservation Tubes (Thermo Scientific, catalog number: 374081)
Poly-Prep Chromatography Columns (Bio-Rad, catalog number: 7311550)
Econo-Pac Chromatography Columns (Bio-Rad, catalog number: 7321010)
SnakeSkin Dialysis Tubing (Thermo Fisher Scientific, catalog number: 68700)
SnakeSkin Dialysis Clips (Thermo Fisher Scientific, catalog number: 68011)
Staples 2” Binder Clips, Large (Staples, catalog number: 10669)
5 ml Serological pipette (Thermo Fisher Scientific, catalog number: 13-678-11D)
10 ml Serological pipettes (Thermo Fisher Scientific, catalog number: 13-678-11E)
50 ml Falcon tube (Cell star, catalog number: 22761)
Precision Plus Protein Kaleidoscope 500 ml (Bio-Rad, catalog number: 161-0375)
Any kD Mini-PROTEAN TGX Stain-Free Protein Gels, 12-well, 20 μl (Bio-Rad, catalog number: 4568125)
2× Laemmli Sample Buffer (Bio-Rad, catalog number: 1610737)
Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 2-gel (Bio-Rad, catalog number: 1658005)
2-Mercaptoethanol, 10 ml (Sigma-Aldrich, catalog number: M6250)
Coomassie Brilliant Blue R-250 Dye (Thermo Fisher Scientific, catalog number: 20278)
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.
Mr. Frosty (Thermo Scientific, catalog number: 5100-0001)
Ni-NTA Agarose (QIAGEN, catalog number: 30230). Store at 2°C to 8°C
DMSO (Millipore Sigma, catalog number: 41640)
Opti-MEM Medium (Thermo Fisher Scientific, catalog number: 11-058-021)
Tris (MP, catalog number: 103133)
NaCl (Fisher, catalog number: S271-10)
Glycerol (VWR, catalog number: BHD 1172-1LP)
Sucrose (Fisher, catalog number: BP-220-1)
Imidazole (Thermo Fisher Scientific, catalog number: 03196-500)
Liquid nitrogen (Arc gases)
Purify Buffer (see Recipes)
Elution Buffer (see Recipes)
Dialysis Buffer (see Recipes)
Stain Buffer for SDS PAGE (see Recipes)
Destain Buffer for SDS PAGE (see Recipes)

Equipment

Fisherbrand Shaker Flasks, Plain Bottom, Vented (Thermo Fisher Scientific, catalog number: PBV12-5). Store at room temperature
4 L Beaker (Thermo Fisher Scientific, catalog number: 02-555-25K)
Forma Steri-Cycle i160 CO₂ Incubator (Thermo Fisher Scientific, Forma, catalog number: 51030301)
CO₂ Resistant Shaker (Thermo Fisher Scientific, catalog number: 88881101)
Biological safety cabinet (Labguard, Class II, Type A2)
Precision Water Bath GP 15D–5 L and 10 L (Thermo Scientific, catalog number: TSGP15D)
Magnetic stir bar (Thermo Fisher Scientific, catalog number: 14-512-136)
Centrifuge (Sorvall, model: RC-5B)
Centrifuge (Eppendorf, model: 5810 R)
Centrifuge (Thermo, model: Sorvall Legend Micro 21R)
Freezer (Thermo Scientific Revco RLE60086A -86 °C)
Cryogenic dewar (Cole Parmer)
Mini-PROTEAN^® Tetra Vertical Electrophoresis Cell for Mini Precast Gels (Bio-Rad, Catalogue number: 1658004)

Procedure

English

中文翻译

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

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.
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.