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Jun 2020
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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   

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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), SARS-CoV (SARS-CoV), Coronavirus (冠状病毒), COVID-19 (2019冠状病毒病), Antigen (抗原), Immunoassay (免疫测定), Halo-tag (Halo-tag), Spike protein (纤突蛋白), Antibody (抗体), 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

  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 C02 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

  1. Establishment of the Expi293 Cell Line (Thermo Fisher Scientific)

    1. Remove a cell aliquot from liquid nitrogen.

    2. Immediately hand thaw the cells and place them in a 37°C water bath. Once thawed, swirl the tube gently without submerging completely until only a small amount of ice remains.

    3. Spray hands with 70% ethanol and gently rub the cell vial to decontaminate before transferring into the laminar flow hood.

    4. Use a serological pipette to transfer all tube contents into a plain bottom, vented Fisherbrand Shaker Flask, prewarmed with 30 ml of Expi293 Expression Medium.

    5. Incubate cells at 37°C with ≥ 80% relative humidity and 8% CO2. Set shaking speed to 125 RPM for a 125 ml shaker flask.

    6. Passage cells when the cell density reaches 1 × 106-3 × 106 cell/ml.

      Note: This usually occurs 4-6 days post-thaw.

    7. Proceed to transfection once the cell density reaches approximately 3 × 106-5 × 106 viable cells/ml and cell viability is ≥ 95%.

      Note: To cryopreserve cells for future use, grow the cell culture to 3 × 106-5 × 106 viable cells/ml and centrifuge them at 300 × g for 5 min.

      1. Discard the supernatant, add Expi293 Expression medium with 10% DMSO, and gently resuspend the cells by pipetting.

      2. Dilute the cells to 1 × 107 viable cells/ml and pipet 1 ml aliquots into cryopreservation tubes and freeze with a controlled-rate freezing apparatus at -80°C freezer. After 24 h, transfer to cryogenic dewar for long-term storage and future use. Allow cells to recover in culture for two more passages post-thaw before transfecting.

      Note: For general cell maintenance, passage cells at 0.5 × 106 cell/ml when they reach a density of 3 × 106-5 × 106 cell/ml. Growing past 5 × 106 cell/ml is not recommended.


  2. Transfection (30 ml)

    1. Dilute a total of 75 × 106 cells to a final density of 3 × 106 cell/ml with 25 ml of prewarmed Expi293 Expression Medium in a 125 ml shaker flask.

    2. Dilute 25 μg of RBD expression plasmid DNA in 1.5 ml of Opti-MEM Medium.

    3. Dilute 80 μl of ExpiFectamine 293 Reagent in 1.4 ml of Opti-MEM Medium. Incubate the solution at room temperature for 5 min.

    4. Add diluted plasmid DNA to the solution containing ExpiFectamine 293 Reagent and incubate at room temperature 10-20 min. The volume should be approximately 3 ml.

    5. Transfer 3 ml of the solution into a shaker flask and incubate cells at 37°C with ≥ 80% relative humidity and 8% CO2.

    6. Eighteen to twenty-two hours post-transfection, add 150 μl of ExpiFectamine 293 Transfection Enhancer 1 and 1.5 ml of ExpiFectamine 293 Enhancer 2 to the shaker flask. Incubate cells for up to 5 days post-transfection.

      Note: The procedure can be scaled up proportionally for larger transfections. Cell viability should be above 50% on day 5.


  3. Harvest and Dialysis

    1. Transfer the cell culture to a 50 ml Falcon tube and centrifuge at 3,000-5,000 × g for 5 min at 25°C (Figure 1).



      Figure 1. Schematics of harvesting cell culture and dialysis of supernatant


    2. Transfer the supernatant into a fresh 50 ml Falcon tube and keep it on ice.

    3. Prepare 3 L of dialysis buffer.

    4. Use about 7-8 inches of SnakeSkin Dialysis Tubing and SnakeSkin Dialysis Clips to transfer 30 ml of harvested supernatant into the dialysis buffer.

      Note: Hydrate the membrane with the dialysis buffer before transferring the supernatant.

    5. Place the sealed snakeskin tubing and a magnetic stir bar in a 4-L beaker.

    6. Place the beaker on a magnetic stirrer and allow buffer exchange at 4°C overnight.

    7. Transfer the buffer-exchanged supernatant into a 50 ml Falcon tube.

    Note: Upon harvesting, it is possible to evaluate the success of transfection and protein expression before the affinity chromatography step by SDS-PAGE with the cell culture supernatant (optional). To perform this:

    1. Mix 50 µl of 2× Laemmli Sample Buffer with 2-mercaptoethanol and 50 µl of cell culture supernatant and boil the sample at 95°C for 5 min.

    2. Load 8-15 μl of the reduced sample onto the SDS-PAGE.

    3. Run sample at 170 v in the Mini-PROTEAN Tetra Vertical Electrophoresis Cell for 35 min and visualize the bands after Coomassie staining using the manufacturer’s protocol.


  4. Immobilized metal affinity chromatography

    1. Take 0.5 ml of Ni-NTA resin in a poly-prep chromatography column and equilibrate with 5 ml of purifying buffer in 1 ml increments. Close the column and resuspend the resin in 1 ml of purifying buffer.

      Note: Proportionally adjust the amount of resin needed for larger transfections.

    2. Transfer the equilibrated resin into a 50 ml Falcon tube containing the buffer exchanged supernatant.

    3. Incubate the resin with the supernatant on a rocking shaker for 1 h at 4°C.

    4. Remove the 50 ml Falcon tube from the rocker and place it on a stand. Allow the resin to settle for 20 min.

    5. Transfer the supernatant into a new 50 ml Falcon tube without disturbing the resin.

    6. Transfer the resin directly onto the bed of the poly-prep chromatography column.

    7. Wash the resin with 6 ml of purifying buffer in 1 ml increments by pipetting on the chromatography column wall.

      Note: Allow the buffer to flow through the column completely before adding another ml of wash buffer.

    8. Elute the protein by adding 200 μl of elution buffer onto the column wall for a total of 7 fractions.

    9. Quantify the protein by measuring the absorbance of the fraction at 280 nm.

    10. Assess the protein purity by SDS PAGE run under reducing conditions (Figure 2).



      Figure 2. SDS-PAGE analysis of purified spike RBD proteins


  5. Storage

    Purified proteins can be stored at 4°C for a few weeks. For long-term storage, protein samples can be aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C. Frozen protein samples can be quickly hand thawed before use.

Recipes

  1. Purify buffer

    50 mM Tris pH 8

    105 mM NaCl

    10% glycerol

    10% sucrose

  2. Dialysis buffer

    50 mM Tris-HCl, pH 8

    100 mM NaCl

  3. Elution buffer

    50 mM Tris pH 8

    105 mM NaCl

    10% glycerol

    10% sucrose

    300 mM imidazole

  4. Stain Buffer for SDS PAGE (1 L)

    500 ml deionized water

    100 ml methanol

    100 ml glacial acetic acid

    3 g brilliant blue

  5. Destain Buffer for SDS PAGE (1 L)

    500 ml deionized water

    400 ml methanol

    100 ml glacial acetic acid

Notes

Codon-optimized nucleotide sequences encoding the RBDs of SARS-CoV-1 (318-514 aa, P59594), SARS-CoV-2 (331-528 aa, QIS60558.1), OC43 (329-613 aa, P36334.1), HKU-1 (310-611 aa, Q0ZME7.1), 229E (295-433 aa, P15423.1), and NL63 (480-617 aa, Q6Q1S2.1) are available in GenBank under the accession codes MT649401, MT649402, MT649403, MT649404, MT649405, and MT649406. The genes encoding the proteins above were cloned between KpnI and XhoI sites of the mammalian expression plasmid pαH. The mammalian expression plasmids will also be made available by the authors from the plasmid repository (Addgene).

Acknowledgments

This work was funded by the University of North Carolina School of Medicine and NCI U54 CA260543-01 (L.P. and A.D). This protocol was adapted with minor modification from previous study published by Premkumar et al. (2020).

Competing interests

The authors have declared no competing interest. This protocol was adapted with minor modification from previous study published by Premkumar et al. (2020).

References

  1. Grifoni, A., Weiskopf, D., Ramirez, S. I., Mateus, J., Dan, J. M., Moderbacher, C. R., Rawlings, S. A., Sutherland, A., Premkumar, L., Jadi, R. S., Marrama, D., de Silva, A. M., Frazier, A., Carlin, A. F., Greenbaum, J. A., Peters, B., Krammer, F., Smith, D. M., Crotty, S. and Sette, A. (2020). Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell 181(7): 1489-1501 e1415.
  2. Mateus, J., Grifoni, A., Tarke, A., Sidney, J., Ramirez, S. I., Dan, J. M., Burger, Z. C., Rawlings, S. A., Smith, D. M., Phillips, E., Mallal, S., Lammers, M., Rubiro, P., Quiambao, L., Sutherland, A., Yu, E. D., da Silva Antunes, R., Greenbaum, J., Frazier, A., Markmann, A. J., Premkumar, L., de Silva, A., Peters, B., Crotty, S., Sette, A. and Weiskopf, D. (2020). Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans. Science 370(6512): 89-94.
  3. 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.
  4. Puhl, A. C., Fritch, E. J., Lane, T. R., Tse, L. V., Yount, B. L., Sacramento, C. Q., Fintelman-Rodrigues, N., Tavella, T. A., Maranhao Costa, F. T., Weston, S., Logue, J., Frieman, M., Premkumar, L., Pearce, K. H., Hurst, B. L., Andrade, C. H., Levi, J. A., Johnson, N. J., Kisthardt, S. C., Scholle, F., Souza, T. M. L., Moorman, N. J., Baric, R. S., Madrid, P. B. and Ekins, S. (2021). Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine, and Pyronaridine: In Vitro Activity against SARS-CoV-2 and Potential Mechanisms. ACS Omega 6(11): 7454-7468.

简介

[摘要] SARS-CoV-1和2病毒突触蛋白的重组受体结合结构域(RBD)是检测人类病毒特异性抗体的可靠抗原。我们和其他人已经表明,患者中RBD结合抗体和SARS-CoV-2中和抗体的水平是相关的。在这里,我们报道了哺乳动物细胞中SARS和常见的HCoVs中正确折叠的RBD蛋白的表达和纯化。RBD蛋白产生带有可切割标签,用于从细胞培养基中进行亲和纯化,并支持多种免疫测定平台和药物发现的努力。


图形摘要:

大量生产用于诊断和药物发现的病毒性穗状RBD


[背景]冠状病毒刺突蛋白的受体结合域(RBD)对于病毒附着,融合和进入至关重要。它也是抗体反应以及进入抑制剂和疫苗开发的主要目标。该RBD小号2003年至2019年SARS的COVS和四种常见的风土人情COVS不易保存,代表用于检测有前途的抗原病毒的特异性抗体人。我们最近表明,SARS-CoV的-2的RBD是高度敏感和特异的检测抗体9天后的症状的发作(Premkumar等人。,2020)。RBD的水平-结合抗体在人血清强烈与患者的SARS-COV-2和效价相关。因此,基于RBD-血清学检测是有吸引力的,以识别个人和环境危险因素为严重的疾病,并监测SARS-COV-2在社区传播。据报道,先前对普通人类地方性冠状病毒(229E,NL63,OC43和HKU1)的免疫增强了对SARS-CoV-2的炎症反应(Grifoni等,2020 ;Mateus等,2020)。在这里,我们介绍了用于表达和纯化2003年和2019年SAR CoVs和四种常见地方性人类CoVs的RBD的详细分步方法,用于血清学检测和抑制剂筛选(Premkumar等,2020和Puhl等, 20 2 1 )。该技术允许小号生产可靠性框图的稠合至TEV蛋白酶可切割自标记蛋白质(HaloTag )在N -末端和一个双Strep标签和在C末端His标签。标签我们重新设计,以辅助亲和纯化和定向抗原的捕获在固体载体上结合的StrepTactin ,链霉或镍-次氮基三乙酸。当前的方案利用哺乳动物表达系统(Expi293),只需一个亲和纯化步骤,即可在5-7天内从少量细胞培养物中产生毫克量的重组RBD 。

关键字:SARS-CoV-2, SARS-CoV, 冠状病毒, 2019冠状病毒病, 抗原, 免疫测定, Halo-tag, 纤突蛋白, 抗体, 抑制剂的筛选, 药物研发



材料和试剂


1.冷冻保存管(热小号系统求解,目录号:374081)     

2. Poly-Prep色谱柱(Bio-Rad,目录号:7311550)     

3. Econo- Pac色谱柱(Bio-Rad,目录号:7321010)     

4. SnakeSkin透析管(Thermo Fisher Scientific,目录号:68700)     

5. SnakeSkin透析夹(Thermo Fisher Scientific,目录号:68011)     

6.大号订书钉2英寸装订夹(订书钉,货号:10669)     

7. 5毫升血清移液管(Thermo Fisher Scientific,目录号:13-678-11D)     

8. 10毫升血清移液管(Thermo Fisher Scientific,目录号:13-678-11E)     

9. 50 ml猎鹰管(Cell star,目录号:22761)     

10.精密加蛋白万花筒500米升(Bio-Rad公司,目录号:161-0375) 

11.任何kD的迷你-PROTEAN TGX染色免费蛋白凝胶,12 -好,20 μ升(Bio-Rad公司,目录号:4568125) 

12. 2 × Laemmli样品缓冲液(Bio-Rad,目录号:1610737) 

13.用于微型预制凝胶的Mini-PROTEAN Tetra垂直电泳池,2凝胶(Bio-Rad,目录号:1658005) 

14. 2-巯基乙醇,10μM升(Sigma-Aldrich公司,目录号:M6250) 

15.考马斯亮蓝R-250染料(热˚F isher科学,Ç atalog号码:20278) 

16. Expi293表达系统套件(Thermo Fisher Scientific,目录号:A14635) 

注意:将细胞储存在2 °C至8°C的液氮和其他试剂中。


17.雾先生(热小号系统求解,目录号:5100-0001) 

18.Ni -NTA琼脂糖(QIAGEN,目录号:30230)。储存在2 °C至8°C 

19. DMSO (Millipore Sigma,目录号:41640) 

20. Opti-MEM培养基(Thermo Fisher Scientific,目录号:11-058-021) 

21. Tris (MP,目录号:103133) 

22. NaCl (Fisher,目录号:S271-10 ) 

23.甘油(VWR,目录号:BHD 1172-1LP ) 

24.蔗糖(Fisher,目录号:BP-220-1 ) 

25.咪唑(Thermo Fisher Scientific,目录号:03196-500) 

26.液氮(电弧气体) 

27.纯化缓冲液(请参阅食谱) 

28.洗脱缓冲液(请参见配方) 

29.透析缓冲液(请参见配方) 

30.用于SDS PAGE的染色缓冲液(请参见配方) 

31.用于SDS PAGE的去污缓冲液(请参阅食谱) 



设备


Fisherbrand摇瓶,平底,通风孔(Thermo Fisher Scientific,目录号:PBV12-5)。室温保存
4 L烧杯(Thermo Fisher Scientific,目录号:02-555-25K)
FORMA免缝-CYCLE I160 CO 2培养箱中(热Fisher Scientific公司,FORMA,目录号:51030301 )
防CO 2摇床(Thermo Fisher Scientific,目录号:88881101 )
生物安全柜(Labguard ,II类,A2型)
精密水浴箱GP 15D–5 L和10 L(Thermo Scientific,目录号:TSGP15D)
磁力搅拌棒(Thermo Fisher Scientific,目录号:14-512-136)
离心机(Sorvall ,型号:RC-5B)
离心机(Eppendorf ,型号:5810 R)
离心机(Thermo ,型号:Sorvall Legend Micro 21R)
冰箱(Thermo Scientific Revco RLE60086A -86 °C)
低温杜瓦瓶(Cole Parmer)
迷你PROTEAN ®四垂直电泳槽的小型预制胶(Bio-Rad公司,目录编号:1658004)


程序


建立了换货Expi293细胞株(赛飞世尔科技)
从液氮中取出细胞等分试样。
马上用手解冻的细胞,并将它们在37 ℃水浴。一旦解冻,涡旋管轻轻而不完全浸没,直到仅一个冰残余的少量。
喷用70%乙醇的手和轻轻擦在转移到前细胞的小瓶以净化所述层流罩。
使用血清移液器将所有试管内容物转移到一个平底,通风的Fisherbrand摇瓶中,并用30 ml Expi293表达培养基预热。
在37°C ,相对湿度≥80%和8%CO 2的条件下孵育细胞。设定振荡速度于125 RPM下一个125毫升小号haker ˚F拉斯克。
当细胞密度达到1 × 10 6 -3 × 10 6细胞/ ml时传代细胞。
注意:这通常在解冻后4-6天发生。


进行到转染一次的细胞密度达到大约3 × 10 6 -5 × 10 6活细胞/ ml和细胞活力是≥95% 。
注意:要冷冻保存细胞以备将来使用,请将细胞培养物生长至3 × 10 6 -5 × 10 6活细胞/ ml,并以300 × g离心5分钟。                         

弃去上清液,加入含10%DMSO的Expi293表达培养基,然后通过移液轻轻重悬细胞。
将细胞稀释至1 × 10 7活细胞/毫升,然后将1毫升等分试样吸取至冷冻保存管中,并用控制速率的冷冻设备在-80 ° C的冰箱中冷冻。24小时后,转移到低温杜瓦长-长期储存和日后使用。在转染前将细胞解冻后再培养两次,使其恢复培养。
注:对于一般的细胞维持,传代细胞以0.5 × 10 6个细胞/ ml,当他们到达的3的密度× 10 6 -5 × 10 6个细胞/ ml。不建议超过5 × 10 6细胞/ ml。


转染(30毫升)
共75个的稀释× 10 6细胞的3的最终密度× 10 6个细胞/ ml用25ml预热Expi293表达培养基在一个125毫升摇瓶中。
稀25 μ克于1.5ml的Opti-MEM培养基的RBD表达质粒DNA。
稀80微升的ExpiFectamine在1.4毫升的Opti-MEM培养基的293试剂。孵育所述在室温下的溶液5分钟。
将稀释的质粒DNA添加到含有ExpiFectamine 293试剂的溶液中,并在室温下孵育10-20分钟。在V olume应该约3μm升。
转移3米升的所述溶液加入到一个摇瓶,并培育的细胞在37℃下用≥80%相对湿度和8%CO 2 。
18到20 -两个ħ我们的小号转染后,加入150微升的ExpiFectamine 293转染增强器1和1.5毫升ExpiFectamine 293增强器2的摇瓶。转染后最多孵育5天。
注:该程序可以按比例高达比例较大的转小号。第5天的细胞活力应高于50%。


收获与透析
转移的细胞培养至50ml的Falcon管中并离心以3000 - 5000 ×克5分钟,在25 ° C(图1)。






图1.收集细胞培养物和上清液透析的示意图


转移的上清液到新的50ml Falcon管中,并保持它在冰上。
准备3 L的透析缓冲液。
约7-8英寸使用蛇皮透析管中,蛇皮透析剪辑传送30米升收获上清液到透析缓冲液中。
注意:转移上清液之前,用透析缓冲液对膜进行水合。


放置在密封的蛇皮管,并在4磁搅拌棒-大号烧杯中。
将烧杯放在磁力搅拌器上,并于4 °C过夜交换缓冲液。
传送缓冲器-交换上清液至50ml的Falcon管中。
注:在收割,有可能评估转染和蛋白表达的成功的亲和层析步骤之前,通过SDS-PAGE用的细胞培养物上清液(可选)。要执行此操作:


将50 µl 2 × Laemmli样品缓冲液与2-巯基乙醇和50 µl细胞培养上清液混合,然后在95°C下煮沸样品5分钟。
加载8-15 μ升的缩小样品到SDS-PAGE。
在Mini-PROTEAN Tetra垂直电泳池中以170 v运行样品35分钟,并按照制造商的规程对考马斯染色后的条带进行可视化。


固定金属亲和色谱
将0.5 ml的Ni-NTA树脂放入多制备色谱柱中,并以5 ml的纯化缓冲液以1 ml的增量进行平衡。关闭柱,重悬在1米树脂升纯化缓冲液中。
注:按比例调整需要较大的转染树脂的量小号。


转移的平衡树脂成一个50米升含有Falcon管的缓冲液更换上清液。
孵育与树脂的上清液上在4℃下1个小时在摇床上。
除去的从摇杆50毫升Falcon管并放置其上的支架。使树脂沉降20分钟。
将上清液转移到新的50 ml的Falcon管中,而不会干扰树脂。
将树脂直接转移到制备型色谱柱的床上。
通过移液至色谱柱壁上,以6 ml的纯化缓冲液以1 ml的增量洗涤树脂。
注:允许缓冲器到完全加入另一米之前流过柱升洗涤缓冲液中。


洗脱的加入200蛋白μ升洗脱缓冲液到塔壁,共7级分。
通过测量级分在280 nm处的吸光度来定量蛋白质。
通过在还原条件s下运行的SDS PAGE评估蛋白质纯度(图2)。






图2.纯化的尖峰RBD蛋白的SDS-PAGE分析


贮存
纯化的蛋白质可以在4°C下保存数周。为了长期保存,可以将蛋白质样品等分,在液氮中快速冷冻,然后保存在-80°C下。冷冻的蛋白质样品可以在使用前快速进行手动解冻。


菜谱


净化缓冲液
50 mM Tris pH 8


105毫米氯化钠


10%甘油


10%蔗糖


透析缓冲液
50 mM Tris-HCl,pH 8


100毫米氯化钠


洗脱缓冲液
50 mM Tris pH 8


105毫米氯化钠


10%甘油


10%蔗糖


300 mM咪唑


用于SDS PAGE的染色缓冲液(1 L)
500毫升去离子水


100毫升甲醇


100毫升冰醋酸


3克艳蓝


用于SDS PAGE的去污缓冲液(1 L)
500毫升去离子水


400毫升甲醇


100毫升冰醋酸


注意小号


编码SARS-CoV-1(318-514氨基酸,P59594),SARS-CoV-2(331-528氨基酸,QIS60558.1),OC43(329-613氨基酸,P36334.1)的RBD的密码子优化核苷酸序列GenBank中提供了HKU-1(310-611 aa,Q0ZME7.1),229E(295-433 aa,P15423.1)和NL63(480-617 aa,Q6Q1S2.1),登录号为MT649401,MT649402 ,MT649403,MT649404,MT649405和MT649406。编码上述蛋白质的基因被克隆到哺乳动物表达质粒pαH的KpnI和XhoI位点之间。作者也可以从质粒储存库(Addgene )获得哺乳动物表达质粒。




致谢


这项工作由北卡罗来纳大学医学院和NCI U54 CA260543-01 (LP和AD)资助。


利益争夺


作者宣称没有竞争利益。


参考


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