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Heterologous Expression and Purification of SARS-CoV2 Nucleocapsid Protein

AG Ankur Garg
LL Lihong Liu
DH David D. Ho
LJ Leemor Joshua-Tor

Published: Aug 5, 2020 DOI: 10.21769/BioProtoc.3720 Views: 1620

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Abstract

This protocol describes a step by step method for heterologous expression of SARS-CoV2 Nucleocapsid (N) protein in Escherichia coli. Moreover, this protocol includes steps to purify the N protein to high purity and homogeneity. Thus, purified protein can be used for ligand binding assays and other biochemical experiments.

Keywords: COVID-19 search SARS CoV2 search Nucleocapsid protein search Recombinant expression search Protein purification search

Background

Since its detection in late 2019 in Wuhanm, China, SARS-CoV2 infections have been rampant around the world (Wu et al., 2020). In an effort to follow the course of early infections, the nucleocapsid protein (N) was used along with the spike protein (S) in serological assays to monitor the course of early infections in the New York area. N is one of the most highly expressed viral protein, and therefore a good target to follow. To facilitate these assays, a purification protocol was developed for N and used successfully in these studies.

Materials and Reagents

  1. Syringe (VWR International, catalog number: BD309653 )
  2. 0.2 μm Syringe filters (VWR International, catalog number: 28196-368 )
  3. Econo-Column 2.5 x 10 cm (Bio-Rad, catalog number: 737-4251 )
  4. HiTrap Heparin HP column 5 ml (GE Healthcare, catalog number: 17040703 )
  5. Amicon Ultra-15 Centrifugal Filter Units 30 kD MWCO (Millipore, catalog number: UFC803096 )
  6. HiLoad 16/600 Superdex200 pg column (GE Healthcare, catalog number: 17106901 )
  7. 96-well Uniplate (Fisher Scientific, catalog number: 09-003-36 )
  8. Culture plates (Thermo Fisher Scientific, catalog number: 0 8-757-100D )
  9. Culture flasks (Thermo Fisher Scientific, catalog number: 09-552-70 )
  10. E. coli Rosetta 2 (DE3) chemically competent cells (Novagen, catalog number: 71400-3 )
  11. pET28a(+)_Nucleocapsid plasmid [pET28a(+) vector (Novagen, catalog number: 69864-3 ) with SARS CoV2 Nucleocapsid encoding gene fused to an in frame C-terminal AAALE linker and a 6xHis tag]
    Note: pET28a(+)_nucleocapsid plasmid is available upon request from the corresponding author.
  12. Agar (Fisher Scientific, catalog number: BP1423-500 )
  13. Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
  14. Tryptone (Fisher Scientific, catalog number: BP1421-2 )
  15. Di-potassium hydrogen phosphate (K2HPO4) (Sigma-Aldrich, catalog number: 60353 )
  16. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: 92214 )
  17. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  18. 1,000x Kanamycin stock solution (50 mg/ml) (Gold Biotechnology, catalog number: K-120-25 )
  19. 1,000x Chloramphenicol stock solution (34 mg/ml) (Gold Biotechnology, catalog number: C-105-5 )
  20. 1 M IPTG (IsoPropyl-1-Thio-B-D-Galactopyranoside) (Gold Biotechnology, catalog number: I2481C50 )
  21. Tris (Gold Biotechnology, catalog number: T-095-1 )
  22. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  23. β-Mercaptoethanol (β-Me) (Sigma-Aldrich, catalog number: M6250-250ML )
  24. 100% Glycerol (Fisher Scientific, catalog number: G33-4 )
  25. TurboNuclease (Accelagen, catalog number: N0103M )
  26. Protease inhibitor cocktail (2 μM Pepstatin, 6 μM Leupeptin, 1 μM PMSF, and 2 mM Benzamidine) (Sigma-Aldrich, catalog numbers: 11524488001 , 11017128001 , p7626-25 , B6506-25 )
  27. Polyethylenimine (PEI) (Acros Organics, catalog number: AC178571000 )
  28. Ni-NTA Agarose resin (Qiagen, catalog number: 30250 )
  29. Bradford assay dye reagent concentrate (Bio-Rad, catalog number: 500-0006 )
  30. Imidazole (Affymetrix, catalog number: 17525 )
  31. ATP (P212121, catalog number: CI-00015-25G )
  32. 12% Mini-PROTEAN TGX Precast protein gel, 15-well (Bio-Rad, catalog number: 4561046 )
  33. 4x Laemmli protein sample buffer for SDS-PAGE (Bio-Rad, catalog number: 1610747 )
  34. InstantBlue Ultrafast protein gel stain (VWR Scientific, catalog number: 95045-070 )
  35. Liquid Nitrogen
  36. Luria-Bertani (LB) medium (see Recipes)
  37. Terrific Broth (TB) medium (see Recipes)
  38. Resuspension buffer (see Recipes)
  39. Ni-NTA buffers
    Wash buffer (see Recipes)
    Elution buffer 1 (see Recipes)
    Elution buffer 2 (see Recipes)
  40. Heparin buffers
    Heparin buffer A (see Recipes)
    Heparin buffer B (see Recipes)
    Heparin dilution buffer (see Recipes)
  41. Size exclusion (SEC) buffer (see Recipes)
  42. 10x SDS-PAGE running buffer (see Recipes)

Equipment

  1. Nalgene PPCO Centrifuge bottles (1 L) (Fisher Scientific, catalog number: 05-562-25 )
  2. Ultracentrifuge tubes (Polycarbonate bottle, 38 x 102 mm, 70 ml) (Beckman Coulter Life Sciences, catalog number: 355655 )
  3. Beaker (Sigma-Aldrich)
  4. Magnetic stirrer (Sigma-Aldrich)
  5. Rocker (IBI Scientific)
  6. Sonicator (12.7 mm tip) (QSonica LLC)
  7. Incubator (at 37 °C)
  8. Incubator shaker (Eppendorf New Brunswick Innova)
  9. Centrifuge (Beckman Coulter Life Sciences, model: J6-MI )
  10. Rotor (Beckman Coulter Life Sciences, model: JS 4.3 )
  11. Ultracentrifuge (Beckman Coulter Life Sciences)
  12. Ultracentrifuge Rotor (Beckman Coulter Life Sciences, model: Ti45 )
  13. Mini-PROTEAN® Tetra Vertical Electrophoresis Cell system (Bio-Rad, catalog number: 165-8005 )
  14. AKTA pure FPLC system (GE Healthcare)
  15. Cold room
  16. UV-VIS spectrophotometer (Thermo Fisher Scientific, model: NanoDrop1000 )

Procedure

Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Heterologous expression system > Escherichia coli

Microbiology > Microbial biochemistry > Protein > Isolation and purification

Molecular Biology > Protein > Expression

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