Production of recombinant HCV E2 ectodomain in a Baculovirus expression system

J. Gómez-Gutiérrez^{1, #}, F. Gavilanes¹ and B. Yélamos^1, *^{, #}

¹Dept. Biochemistry and Molecular Biology, Complutense University of Madrid, Madrid, Spain

*For correspondence: mbyelamo@ucm.es

^#Contributed equally to this work

[Abstract]

In this protocol we describe the production and purification of the ectodomain of the E2₆₆₁ envelope protein of the Hepatitits C virus, which plays a fundamental role in the entry of the virus into the host cell. This protein has been expressed in both prokaryotes and eukaryotes systems but in small quantities or without native protein characteristics. In our case, we use the Baculovirus expression system in insect cells. E2₆₆₁ is secreted into the extracellular medium and purified by means of affinity chromatography a Ni-NTA-column because the protein has a tag of six histidines at its amino terminal end. The purified protein possesses a native-like conformation and it is produced in large quantities, around 5-6 mg per liter.

Keywords: Hepatitis C virus, envelope protein, affinity chromatography, baculovirus expression system.

[Background]

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide [1, 2]. At this moment, there is no vaccine for HCV and recently, new antivirals are used to treat the HCV infection [3]. However, the treatments are expensive and not 100% effective [4]. The HCV envelope glycoprotein E2 is responsible for the interaction with cellular receptors, then it is a major candidate to study the first steps of the infective cycle of the virus. Previous expression systems produce low levels of heterogeneous protein due to glycosylation and aggregation, and it is difficult to distinguish between molecules that undergo productive and non-productive folding [5]. In this protocol, we describe the production of the recombinant ectodomain of E2 tagged with a 6xHis extension at N-terminal end of the protein in a baculovirus/insect cell system. The protein is secreted to the cell supernatant and purified by means of affinity chromatography with a Ni-NTA-Agarose column. The yield of the process was 5–6 mg of protein per litter of media. This protein possesses a native-like conformation. The use of this independent folding domain may contribute to shed light on the biology of HCV and could also be used as a vaccine in the prevention of HCV infection.

Materials and Reagents

1. Tissue culture flasks F75 (75 cm² surface area) (TPP, 90075, Switzerland)

2. Tissue culture flasks F150 (150 cm² surface area) (TPP, 90150, Switzerland)

3. Cell culture flasks F25 (25 cm² surface area) (Corning, 30555, USA)

4. Tissue culture dish 35 mm (Sarstedt, 83.100, USA)

5. Sterile tube 50 ml (Sarstedt, 62.547.254, Germany)

6. Sterile tube 15 ml (Fisher Scientific, 05-539-12, Spain)

7. Serological pipette 10 ml (Sarstedt, 86.1254.001, Germany)

8. Dialysis membrane Spectra/Por® 6 (Spectrum, 734-0646, VWR, Spain)

9. Baculovirus transfer vector pAcGP37A (BD Biosciences, Pharmingen, catalogue number: 21220P)

10. Recombinant transfer vector pAcGP67A-E2₆₆₁ [6]

11. FlashBAC GOLD kit (Oxford Expression Technologies, catalogue number: 100201, England)

12. Insect cell line Spodoptera frugiperda (Sf9) (Oxford Expression Technologies, catalogue number: 100201, England)

13. Insect cell line Trichopulsia ni (High Five) (Tni) (generously donated by PhD J. Pérez-Gil, Dpt. Biochemistry and Molecular Biology, Faculty of Biology, University Complutense of Madrid, Madrid, Spain)

14. Insect-XPRESS™ Protein-free Insect Cell Medium with L-glutamine (Lonza, catalogue number: 12-730Q)

15. Gentamicin (Sigma-Aldrich; Catalogue number: G-1264; USA )

16. TC100 Insect Medium (Lonza, catalogue number: 733-1661)

17. Ni²⁺-nitrilotriacetic acid agarose (Ni-NTA agarose) resin (Qiagen; Catalogue number: 30230; Germany)

18. Tris–Base (Sigma-Aldrich; Catalogue number: T1503; USA)

19. NaCl (Merck; Catalogue number:106404; Germany)

20. Imidazole (Sigma-Aldrich; Catalogue number: 56750; USA)

21. EDTA (Sigma-Aldrich; Catalogue number: 03695; USA)

22. Dodecyl sulfate sodium salt (Merck; Catalogue number: 1.13760.1000; Germany)

23. Glycerol (Sigma-Aldrich; Catalogue number: 5516; USA)

24. Glycine (Sigma-Aldrich; Catalogue number: G8898-1KG; USA)

25. Bromophenol blue (USA Biochemical Corporation; Catalogue number: 12370; USA)

26. 2-mercaptoethanol (Sigma-Aldrich; Catalogue number: M6250; USA)

27. Acrylamide (Sigma-Aldrich; Catalogue number: A8887-500G; USA

28. N,N′-Methylenebis(acrylamide) (Sigma-Aldrich; Catalogue number: M7279-250G; USA)

29. Ammonium persulphate (Sigma-Aldrich; Catalogue number: A3678-100G; USA)

30. N,N,N',N',N'-tetramethylethylenediamine (TEMED) (Bio-Rad, Catalogue number: 1610801, Spain)

31. Coomassie Brilliant Blue R-250 (Sigma-Aldrich; Catalogue number: B8647; USA)

32. Methanol (Sigma-Aldrich; Catalogue number: 322415-1L; USA)

33. Acetic acid (Merck; Catalogue number: 1000063; Germany)

Equipment

1. CO₂ Water-jacketed Incubator (NUAIRE^TM, NU-2700 IR Autoflow Incubator, USA)

2. Laminar flow chamber (Telstar, BV-100, Spain)

3. Laboratory centrifuge (MPW Medical Instruments, MPW-223e, Poland)

4. Upright microscope (Nikon Instruments, IZASA, H550S, Spain)

5. Mini-PROTEAN® Tetra Electrophoresis System (Bio-Rad, 165-8001, USA)

6. UV-Spectrophotometer (Shimazdu; UV-1800; Germany)

Procedure

Generation of recombinant baculoviruses by cotransfection

1. Seed the 35 mm cell culture dishes with 1.5 x 10⁶ Sf9 cells in 2 ml of Insect-XPRESS™ medium with gentamycin at 10 g/ml to form a sub-confluent monolayer.

2. Incubate during 1 hour to allow cell attachment to the plate.

3. Prepare the co-transfection mix of DNA and transfection reagent in a polystyrene tube:

• 100 l of serum free medium (TC100)

• 100 ng of virus DNA from the flashBAC kit

• 500 ng of transfer plasmid (pAcGP67A-E2₆₆₁ recombinant plasmid or, alternatively lacZ positive control transfer vector from the flashBAC kit)

• 1.2 l of baculoFECTIN II from the flashBAC kit

Mix and leave at room temperature for 15 mins.

4. Wash the cell monolayers twice with TC100 medium without serum and then add 1 ml of TC100 medium without serum to each 35 mm dish. If the cells are maintained in serum-free medium it is not necessary to wash the monolayer. In this case, remove and discard 1 ml of medium from the 35 mm dishes.

5. Add the transfection mix (111.2 l) to the 35 mm dish.

6. Incubate overnight at 27 ºC.

7. Add 1 ml of Insect-XPRESS™ medium with gentamicin at 10 g/ml.

8. Incubate at 27 ºC for 4 days.

9. Harvest the culture medium that contain the recombinant virus into a sterile container and store in the dark at 4 ºC. For a long-term storage, save the container at -80 ºC.

Amplification of recombinant viruses

Once the purified recombinant baculovirus is available, it must be amplified in order to obtain a high virus titre. When the infection aims to obtain recombinant protein, a high virus titre is necessary to infect cells with a multiplicity of infection (MOI) of 5-10 pfu/ml-cell.

1. Seed 2x10⁶ Tni insect cells per 25 cm² of surface area and incubate for 1 hour at 27ºC.

2. Add 1 ml of the supernatant containing the recombinant viruses for every 25 cm² of surface area (Flask F25, 1 ml; Flask F75, 3 ml; Flask F150, 6 ml).

3. Incubate for one hour at 27ºC with very slow agitation to maintain the hydration of the cell monolayer with the minimum volume of inoculum.

4. Add the relevant volume of Insect X-Press medium with gentamicin at 10 g/ml to the flask (Flask F25, until 5 ml; Flask F75, until 10 ml; Flask F150, until 30 ml).

5. Incubate at 27ºC for 5 days.

6. Collect the supernatant and centrifuge for 15 min at 1500 rpm.

7. Use the supernatant for a second round of amplification. Usually, two rounds of amplification give a titre of 2∙10⁷ pfu/ml, sufficient for large-scale infection to allow expression of the recombinant protein of interest.

Production and Purification of HCV E2₆₆₁ protein

1. Seed 10 flasks of 150 cm² surface area with 8-10x10⁶ Tni insect cells and incubate for one hour at 27ºC.

2. Add 6 ml of supernatant containing the recombinant E2₆₆₁ baculovirus per flask.

3. Incubate for one hour at 27ºC with very slow agitation to maintain the hydration of the cell monolayer with the minimum volume of inoculum.

4. Add 24 ml of Insect X-Press medium with gentamicin at 10 g/ml per flask.

5. Incubate at 27ºC for 5 days.

6. Collect the supernatants and centrifuge for 15 min at 1500 rpm.

7. Dialyze the supernatant (300 ml) against 20 L (10 Lx2) of 50mM Tris–HCl, pH 8.0, 300 mM NaCl buffer.

8. Load the supernatant onto a 2 ml Ni-NTA-Agarose column previously equilibrated with 50mM Tris–HCl, pH 8.0, 300 mM NaCl buffer.

9. Wash the column with 50mM Tris–HCl, pH 8.0, 300 mM NaCl, 10 mM Imidazol until optical density at 280 nm is below 0.03.

10. Wash the column with 50mM Tris–HCl, pH 8.0, 300 mM NaCl, 30 mM Imidazol until optical density at 280 nm is below 0.03.

11. Elute the E2₆₆₁ protein with 50mM Tris–HCl, pH 8.0, 300 mM NaCl, 200 mM Imidazol collecting fractions of 1-1.2 ml.

12. The protein is detected by measuring the optical density at 280 nm of each fraction.

13. Collect the fractions containing significant amounts of the E2₆₆₁ protein in a single fraction.

14. Dialyze the E2₆₆₁ protein against 50mM Tris–HCl, pH 8.0, 300 mM NaCl buffer.

15. Check the purity of the recombinant E2₆₆₁ protein by SDS-PAGE.

SDS-PAGE (Protein electrophoresis)

1. Prepare the electrophoresis system.

2. Prepare the separating gel by mixing the following quantities:

• 650 l H₂O

• 1.82 ml Tris 1 M pH 8.8

• 2.5 ml acrylamide-bisacrylamide at 30%

• 55 l SDS 10%

• 12 l of 0.075% TEMED

• 14 l 0.02% (w/v) ammonium persulfate

3. Mix with a pasteur pipette and deposite into the gel stand.

4. Prepare the concentrating gel according to the following quantities:

• 2.0 ml acrylamide-bisacrylamide at 4%

• 55 l SDS 10%

• 8 l of 0.075% TEMED

• 8 l of 0.02% (w/v) ammonium persulfate

5. Mix with a pasteur pipette and deposit on top of the separating gel.

6. Add 10 l of application buffer to 20 l of sample.

7. Heat at 95 ºC for 5 mins.

8. Load into the concentrating gel.

9. Develop the electrophoresis at room temperature at 25 mA per gel until the marker reached the end of the gel.

10. Incubate the gel in a Coomassie blue staining solution at room temperature for 10 mins.

11. Incubate the gel in the bleaching solution to detect the proteins.

Recipes

1. Protein electrophoresis application buffer 3x Tris 150 mM pH 7.6, EDTA 6 mM, SDS 3% (w/v), glycerol 30% (v/v), bromophenol blue 0.06% (w/v) and β-mercaptoethanol 15% (v/v).

2. Running electrophoresis buffer: Tris 0.025 M pH 8.3, glycine 0.192 M with 0.1% SDS.

3. Blue staining solution: Coomassie Brilliant Blue R-250 0.3% (w/v), methanol 45% (v/v) and acetic acid 10% (v/v)).

4. Bleaching solution: 7.5% acetic acid (v/v) and 20% methanol (v/v).

Acknowledgments

This work was supported by grant BFU 2006-13033 from the Ministerio de Educación y Ciencia, Spain and the research project SANTANDER/COMPLUTENSE PR26/16-20271.

The authors should use this section to declare any conflicts of interest or competing interests.

References

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