Protein expression and purification

Danio rerio CALHM1 protein expression and purification

1. Danio rerio CALHM1 protein (Accession: XM_017358898.2) was cloned into pEG-BacMam vector with a TEV protease cleavage site and an enhanced green fluorescent protein (eGFP) at N-terminus.
2. Protein expression was performed as described for the standard Bac-to-Mam Baculovirus expression system (Invitrogen). The drCALHM1 bacmid was generated by DH10Bac cells. Sf9 cells grown at 27 °C were transfected with the bacmid. After about 72 h, the first-generation virus (P1) was harvested. According to the efficiency of the virus, P1 was added to a new generation of Sf9 cells at a cell density of 1.5 x10⁶ cells per mL to further amplify the virus. Then the second-generation virus (P2) was collected after 72 h and was used to infect HEK293S cells in Erlenmeyer flasks with 5% CO₂ at 37 °C at a cell density of 2 x10⁶ cells per mL.
3. After 24 hours, add sodium butyrate to the cell culture at a final concentration of 10 mM. Then transfer to 30 °C and continue to incubate for 60 h.
4. Harvest the cells by centrifugation at 800 ×g for 10 min.
5. Resuspend the cells in lysis buffer and disrupt through sonication for 15-20 min (1s pulse on and 3 s pulse off).
6. Collect the membrane by ultracentrifugation at 180,000 ×g at 4 °C for 1 h.
7. Solubilize the precipitate in buffer A and incubate at 4 °C for 3 h.
8. Remove insoluble material by centrifugation at 110,000 ×g at 4 °C for 40 min.
9. Incubate the supernatant by gentle rotation with agarose beads conjugated with anti-GFP nanobody for 2.5 h.
10. Pack the beads by gravity at 4 °C.
11. Wash the beads three times with W1 buffer and W2 buffer of 3-5 column volumes (CV).
12. Wash the beads with 3CV W3 buffer three times for 20 minutes each.
13. Wash the beads with the buffer that changes the concentration of the two detergents in a gradient, and suspend the beads in 3CV buffer B for 1 hour at 4 °C and then wash several times.
14. Elute protein by incubating the beads with TEV protease for ~12 hours at 4 °C.
15. Concentrate the eluate by using a 100-kDa cut-off Centricon (Merck Millipore) and inject into a Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with buffer B.
16. Run the SDS-PAGE to verify the purity of the protein (Fig.1).
17. Pool the pure protein and concentrate to ~9 mg/mL and immediately use for cryo-EM grid preparation.
    
     

Fig.1 SDS-PAGE

Solutions:

1. Lysis buffer:
   20 mM Tris-HCl pH 7.5,
   200 mM NaCl,
   1 mM PMSF.
2. Buffer A:
   1% (w/v) n-dodecyl-β-D-maltoside (DDM, Anatrace),
   0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma-Aldrich), 20 mM Tris-HCl pH 7.5,
   200 mM NaCl,
   1× protease inhibitor cocktail (Roche).
3. Buffer B:
   20 mM Tris-HCl pH 7.5,
   200 mM NaCl,
   0.0063% (w/v) glycol-diosgenin (GDN, Anatrace).
4. W1 buffer:
   20 mM Tris-HCl pH 7.5,
   500 mM NaCl,
   0.025% (w/v) DDM and 0.005% (w/v) CHS.
5. W2 buffer:
   20 mM Tris-HCl pH 7.5,
   200 mM NaCl,
   0.025% (w/v) DDM and 0.005% (w/v) CHS.
6. W3 buffer:
   10 mM adenosine triphosphate (ATP, Macklin), 10 mM MgCl₂,
   20 mM Tris-HCl pH 7.5,
   200 mM NaCl,
   0.025% (w/v) DDM and 0.005% (w/v) CHS.

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