Heterologous Expression and Purification of SARS-CoV2 Nucleocapsid ProteinPreprint   

Abstract

Materials and Reagents

1. 0.2 μm Syringe filters (Nalgene)
2. Econo-Column 2.5 x 10 cm (BioRad)
3. HiTrap Heparin HP column 5 ml (GE Healthcare)
4. Amicon Ultra-15 Centrifugal Filter Units 30 kD MWCO (Millipore)
5. HiLoad 16/600 Superdex200 pg column (GE Healthcare)
6. Collection tubes or 96 well block
7. E. coli Rosetta 2 (DE3) chemically competent cells (Merck, Novagen)
8. pET28a(+)_Nucleocapsid plasmid
9. Agar (Fisher Scientific)
10. LB media
11. TB media
12. 1,000x Kanamycin stock solution (50 mg/ml) (Gold Biotechnology)
13. 1,000x Chloramphenicol stock solution (34 mg/ml) (Gold Biotechnology)
14. 1 M IPTG (IsoPropyl-1-Thio-B-D-Galactopyranoside) (Gold Biotechnology)
15. Tris (Gold Biotechnology)
16. NaCl (Sigma-Aldrich)
17. ß-Mercaptoethanol (ßMe) (Sigma-Aldrich)
18. 100 % Glycerol (Fisher Scientific)
19. TurboNuclease (Accelagen)
20. Protease inhibitor cocktail (2 μM Pepstatin, 6 μM Leupeptin, 1 μM PMSF, and 2 mM Benzamidine) (Sigma-Aldrich)
21. PEI (Acros Organics)
22. Ni-NTA Agarose resin (Qiagen)
23. Bradford assay dye reagent concentrate (BioRad)
24. Imidazole (Affymetrix)
25. ATP (P212121)
26. Liq-N₂
27. Resuspension buffer (see Recipes)
28. Ni-NTA buffers
    Wash buffer (see Recipes)
    Elution buffer 1 (see Recipes)
    Elution buffer 2 (see Recipes)
29. Heparin buffers
    Heparin buffer A (see Recipes)
    Heparin buffer B (see Recipes)
    Heparin dilution buffer (see Recipes)
30. Size exclusion (SEC) buffer (see Recipes)
    

Equipment

1. Beaker
2. Magnetic stirrer
3. Rocker
4. AKTA pure FPLC system (GE Healthcare)
   

Procedure

1. SARS CoV2 N-protein was cloned into pET28a(+) with an in-frame AAALE linker and 6xHis tag at C-terminal.
2. Transform 50 μl of chemically competent E. coli Rosetta 2 (DE3) cells with the 50-100 ng pET28a(+)_Nucleocapsid plasmid and plate the revival mix onto LB agar plate with Kanamycin (Kan) and Chloramphenicol (Cam) antibiotics, and leave it for incubation overnight in 37 °C incubator.
3. Select a single colony from the LB agar plate and inoculate into 100 ml LB media (supplemented with Kan and Cam) at 37 °C at 220 rpm shaking.
4. Inoculate 5-10 L TB media (supplemented with Kan and Cam) with 1% overnight culture and shake at 200 rpm at 37 °C till the cells grow to an OD of 1.5.
5. Induce protein expression by adding IPTG to 0.5 mM final concentration after reducing the culture temperature to 18 °C, for 16-18 h.
6. Harvest the cells by centrifuging culture at 4,000 x g for 20 min, followed by resuspending the pellet in 20 ml resuspension buffer per liter culture. Pellet can be stored frozen in -80 °C for long term storage.
7. Thaw out the pellet in lukewarm water and add 2 U/ml TurboNuclease or Benzonase in the lysate, and lyse the cells by sonication at 100 % amplitude for 4-5 min (3 s pulse on and 6 s pulse off). For large volumes (>150 ml), divide the lysate into two beakers and sonicate separately.
8. Add PEI (final 0.2 %) to the lysate while mixing with a magnetic stirrer for 5 min and ultracentrifuge the lysate for 1 h at 95,000 x g.
9. Filter the cleared lysate through a 0.2 μm syringe filter and add Ni-NTA agarose beads pre-equilibrated in Wash buffer (0.5 ml per liter culture) to the cleared lysate. Let the Ni-NTA beads incubate on a rocker for 1 h in cold room.
   
10. Separate the Ni-NTA beads from the lysate under gravity flow using Econo-column and wash the beads with 20 CV of Wash buffer followed by protein elution with Elution buffer 1 (4 CV) and Elution buffer 2 (4 CV).
11. Measure the protein amount with Bradford method. Run fractions on SDS-PAGE to check the purity of protein (Figure 1).
    
    
    Figure 1. A representative SDS-PAGE for Ni-NTA affinity chromatography for SARS CoV2 N-protein. Different samples collected during Ni-NTA were analyzed on 12% SDS-PAGE. Ni-NTA elutions show most of the protein with some protein still bound to Ni-beads after elution with 500 mM imidazole buffer.
    
    
12. Pool the elution fractions and dilute 1:1 using Heparin dilution buffer to reduce the NaCl concentration to 100 mM. 
13. Connect HiTrap Heparin-HP column to AKTA pure system and equilibrate the column with 5 CV Heparin buffer A before loading the diluted protein on column using the sample pump.
14. Wash the Heparin HP column with 8 CV Heparin buffer A, followed by protein elution using a linear gradient from 10 to 100 % Heparin buffer B over 150 ml. Protein will elute between 60-70 % Heparin buffer B cons. 
15. Collect 1-2 ml fractions in clean tubes and run the peak fractions on SDS-PAGE for analyzing the purity of the protein. A typical Heparin-HP elution chromatogram for N-protein is shown in Figure 2.
    
    
    Figure 2. A representative SDS-PAGE and chromatogram for Heparin HP (5 ml) chromatography for SARS CoV2 N-protein. N-proteins eluted out between 60-70% Heparin buffer B and the fractions covering whole peak (marked with red arrow on top of the peak) were analyzed on 12% SDS-PAGE. Fractions having pure protein were further purified on size exclusion chromatography.
    
    
16. Pool the pure fractions from HiTrap Heparin HP elutions and concentrate it to ~2 ml final volume using 30 kD MWCO Amicon Ultra-15 Centrifugal Filter Units as per manufacturer’s instructions.
17. Equilibrate the HiLoad 16/600 Superdex200 pg column with SEC buffer and inject the concentrated protein using capillary loop followed by eluting it out with SEC buffer at 1 ml/min flow rate. A single peak for the homogenous protein will be observed. 
18. Collect 1 ml fractions in clean tubes and run the peak fractions on SDS-PAGE for analyzing the protein purity (Figure 3).
    
    
    Figure-3 A representative SDS-PAGE and chromatogram for the size exclusion chromatography for SARS CoV2 N-protein. Concentrated protein was loaded on HiLoad 16/600 Superdex200 pg column and fractions covering whole peak (marked with red arrow in chromatogram) were analyzed on a 12% SDS-PAGE. Only pure protein containing fractions (marked on top of the gel) were pooled, concentrated and stored.
    
    
19. Pool the pure protein containing fractions and concentrate using 30 kD MWCO Amicon Ultra-15 Centrifugal Filter Units to desired concentration.
20. Protein aliquot can be flash frozen in Liq-N₂ and stored at -80 °C for storage.
    

Data analysis

1. This section explains how to determine the yield and purity of protein after size exclusion chromatography.
   1. A Spectrophotometer/Nanodrop can be used to measure the absorbance at 280 nm and 260 nm.
   2. The 280 nm absorbance and the correction factor of 0.932 was used to determine the real concentration of N protein (Real cons = A280/0.932). Purified protein shows a 260/280 ratio between 0.55-0.60, which represents that there is no nucleic acid contamination in the protein.
2. The UNICORN 7.4 software (GE Healthcare) was used to visualize the chromatograms and preparing chromatogram figures.
   

Recipes

1. Resuspension buffer
   Tris-25 mM (pH 8.0)
   NaCl-500 mM
   ßMe-2 mM
   Glycerol-5 %
   Imidazole-10 mM
   Protease inhibitor cocktail
2. Ni-NTA buffers
   
   1. Wash buffer
      Tris-25 mM (pH 7.4)
      NaCl-500 mM
      ßMe -2 mM
      Glycerol-5 %
      Imidazole-50 mM
      ATP-1 mM
   2. Elution buffer 1
      Tris-25 mM (pH 7.4)
      NaCl-200 mM
      ßMe -2 mM
      Glycerol-5 %
      Imidazole-250 mM
   3. Elution buffer 2
      Tris-25 mM (pH 7.4)
      NaCl-200 mM
      ßMe -2 mM
      Glycerol-5 %
      Imidazole-500 mM
3. Heparin buffers
   
   1. Heparin buffer A
      Tris-25 mM (pH 7.4)
      NaCl-100 mM
      ßMe -2 mM
   2. Heparin buffer B
      Tris-25 mM (pH 7.4)
      NaCl-1000 m
      ßMe -2 mM
   3. Heparin dilution buffer
      Tris-25 mM (pH 7.4)
      ßMe -2 mM
4. Size exclusion (SEC) buffer
   Tris-25 mM (pH 8.0)
   NaCl-500 m
   ßMe -2 mM
   

Acknowledgements

This work was supported by the Meier & Linnartz Family Foundation. L.J. is an Investigator of the Howard Hughes Medical Institute.

Competing interests

Competing interests

References

1. Wu, F., Zhao, S., Yu, B., Chen, Y. M., Wang, W., Song, Z. G., Hu, Y., Tao, Z. W., Tian, J. H., Pei, Y. Y., Yuan, M. L., Zhang, Y. L., Dai, F. H., Liu, Y., Wang, Q. M., Zheng, J. J., Xu, L., Holmes, E. C. and Zhang, Y. Z. (2020). A new coronavirus associated with human respiratory disease in China. Nature 579(7798): 265-269.