On-Column Folding of HisTag Protein

1. Spin down cells, cell pellet can be stored at -70°C or processed immediately.

2. Resuspend the cell paste from 1 L culture of E. coli expressing (His)6-tagged recombinant protein in 40 ml 20 mM Tris-HCl, pH 8.0.

3. Disrupt the cells with sonication on ice (e.g. 4 × 10 sec.) and centrifuge at high speed for 10 min. at + 4 °C.

4. The pellet, containing the inclusion bodies, is resuspended in 30 ml cold 2M Urea Wash Buffer (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100 pH 8.0) and sonicate as above.

5. Centrifuge at high speed for 10 min. at + 4 °C.

6. Wash the pellet again with 2M Urea Wash Buffer. Centrifuge at high speed for 10 min. at + 4 °C.

7. Wash the pellet with IB  Wash Buffer (20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100 pH 8.0). Centrifuge at high speed for 10 min. at + 4 °C.

8. Resuspend the pellet in 25 ml Binding Buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0).

9. Stir for 30–60 min. in room temperature

10. Centrifugate 15 min. at high speed, + 4 °C. Collect supernatant.

The optimal concentration of reducing 2-mercaptoethanol (0–5 mM) must be determined experimentally for each individual protein.

11. Wash HiTrap Chelating 1 ml column with 5 ml distilled water using a 5 ml syringe. Load 0.5 ml 0.1 M NiSO4 and continue to wash with 5 ml distilled water. Equilibrate the column with  5–10 ml of Binding Buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0).

12. Load the sample from step 10.

13. Wash the column with 10 ml of Binding Buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0).

14. Wash column with 10 ml of Wash Buffer U (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, 6 M urea, pH 8.0)

15. Refolding using a linear 6–0 M urea gradient, starting with the Wash Buffer U and finishing with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0). A gradient volume of 30 ml or higher and a flow rate of 0.5  ( 0.1 to 1 ) ml/min can be used, while the optimal renaturation rate should be determined experimentally for each protein.

16. Continue to wash with 5 ml of Wash Buffer.

17. Elute the refolded recombinant protein using a 10–20 ml linear gradient starting with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0). and ending with Elution Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0).

2M Urea Wash Buffer: 2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0.

IB Wash Buffer: 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0.
Flow rates: 0.1–1 ml/min, sample loading and refolding 1 ml/min, wash and elution
Binding Buffer: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol, pH 8.0
Washing Buffer U: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 6 M urea, 1 mM  2-mercaptoethanol, pH 8.0
Refolding Buffer A: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol , pH 8.0

Refolding gradient: 30 ml
Elution Buffer B: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0
Elution gradient: 10 ml,  1.0 ml/min
Fraction volumes: 3 ml sample loading, wash and refolding 1 ml elution