His-TEV-Tat-NRF2 Purification

1. Transform Rosetta2(DE3)pLysS (EMD) or BL21(DE3)pRare2 pLysS with plasmid

2. Pick a few colonies and grow them in 80 ml LB/K/C, at 37 oC

3. Add 10 ml of starter culture to 1 liter LB/K/C, at 37 °C until OD600nm reached 0.6.

4. Add 1 M IPTG to final conc of 1 mM, grow for 4 h at 37 oC.

5. Spin down cells, add Lysis buffer (50 mM Tris–HCl, pH 8.0, 400 mM NaCl, 5% glycerol, 0.1 mM EDTA, 2 mM tris(2-carboxyethyl)phosphine (TCEP) or beta-ME). Add at a ratio of 10 ml of lysis buffer per 1 g of wet weight E. coli.

6. Freeze and thaw once.

7. Homogenize or sonicate.

8. Spin at 26,000g for 30 min at 4 °C.

9. Wash inclusion bodies in the pellet twice by resuspension in Inclusion Body Wash Buffer
(50 mM Tris–HCl, pH 7.9, 400 mM NaCl, 5% glycerol, 0.1 mM EDTA, 2 mM tris(2-carboxyethyl)phosphine (TCEP) or beta-ME), 1% Triton X-100).

10. Spin again as step 8.

11. Wash again with Lysis Buffer.

12. Dissolve inclusion bodies in denaturing buffer (50 mM Tris–HCl, 6 M guanidine hydrochloride (GdnHCl), 2 mM TCEP, pH 7.9).

13. Spin at 26,000g for 30 min.

14. Load supernatant (1 ml) to on a 24-ml Superose 6 10/300 GL column.

15. Elute with mobile phase (50 mM Tris–HCl, 3 M GdnHCl, 2 mM TCEP, pH 7.9) at a flow rate of 0.25 ml/min.

16. Solubilize inculsion body in denaturing buffer (10 mM Tris–HCl, 3 M GdnHCl, 0.1 mM dithiothreitol (DTT), pH 7.9), and dilutediluted 20-fold,, i.e., add 10 ul of denatured protein into 190 ul of base buffer (10 mM Tris–HCl, 0.1 mM DTT, pH 7.9) supplemented with specific refolding additives, such as 0.6 M arginine.

Best condition for NRF2 refolding: 25 mM Tris–HCl, 0.6 M arginine, 2 mM TCEP, pH 7.9

17. Incubated for 1 h at 22 °C and then measure absorbance at 320 nm using spectrophotometer or microplate reader. Use one well with water as blank and one well without refolding additive as unfolded control.

Example additives: 50% glycerol, 0.6 M arginine, 0.3 M arginine, sodium taurodeoxycholate, Polyethylene glycol, β-mercaptoethanol, dithiotreitol (DTT), dithioerythritol, or cysteine, Glucose, 0.75 M Trehalose, 1 M Glycine betaine, 0.5 M Mannitol, 0.1 M Potassium citrate, mannitol, L-Arginine, potassium citrate, CuCl2, proline, xylitol, NDSB 201, CTAB and K2PO4.

18. Analyse refolding efficiency by analytical SEC (AnSEC). Equilibrate a Shodex Protein KW802.5 column with mobile phase (50 mM Tris–HCl, 250 mM NaCl, 5% glycerol, 0.1 mM DTT, pH 7.9). Load 200 ul of refolded protein (10–40 ug) onto the column and elute protein with mobile phase at a flow rate of 1 ml/min. Monitor at 280 nm (A280nm) and 215 nm (A215nm).

19. Concentrate and desalting using hydrophobic interaction chromatography (HIC).

• Equilibrate a 1-ml Phenyl-Sepharose FF (low substitution) column equilibrated with Buffer A (50 mM Tris–HCl, 2 mM TCEP, pH 7.5, 800 mM ammonium sulfate)
  * Elute protein with phase B (50 mM Tris–HCl, 2 mM TCEP, pH 7.5).

20. Add 2X buffer TEV buffer (100 mM Tris–HCl, pH 7.9, 300 mM NaCl, 1 mM EDTA, and 0.2 mM DTT, 10% glycerol) to adjust to final conc: 50 mM Tris–HCl, pH 7.9, 150 mM NaCl, 0.5 mM EDTA, and 0.1 mM DTT, 5% glycerol) and cleaved with 0.1 M equivalents of His6-tagged TEV protease for 2 h at 22 °C.

21. Pass digested protein through a 1 ml Ni-NTA agarose column, collect flow through.

22. Concentrate protein using spin concentrator.

23. Store protein at -80 oC.