On Column Refolding

Disruption, wash and isolation ofinclusion bodies

Resuspend the cell paste from 100 ml culture of E. coli expressing (His)6-tagged recombinant protein in 4 ml 20 mM Tris-HCl pH 8.0. Disrupt the cells with sonication on ice (e.g. 4 × 10 sec.) and centrifuge at high speed for 10 min. at + 4 °C. The pellet, containing the inclusion bodies, is resuspended in 3 ml cold 2 M urea, 20 mM Tris-HCl, 0.5 MNaCl, 2% Triton™ X-100 pH 8.0 and sonicated as above.Centrifuge at high speed for 10 min. at + 4 °C. Subject the pellet to a second round of urea wash. At this stage the pellet material can be washed once in buffer lacking urea and stored frozen for later processing

Solubilization and sample preparation

Resuspend the pellet in 5 ml 20 mM Tris-HCl, 0.5 M NaCl,5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0. Stir for 30–60 min. in room temperature and centrifugate 15 min. at high speed, + 4 °C. Remove remaining particles by passing the sample through a 0.22 μm or 0.45 μm filter. The optimal concentration of reducing 2-mercaptoethanol (0–5 mM) must be determined experimentally for each individual protein

Preparation of the column

HiTrap™ Chelating 1 ml column is washed with 5 ml distilled water using a 5 ml syringe. Load 0.5 ml 0.1 M NiSO4 andcontinue to wash with 5 ml distilled water. Equilibrate thecolumn with 5–10 ml 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM2-mercaptoethanol pH 8.0.

Purification and refolding

Loading and washing

Load the sample and wash the column with 10 ml 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0. Change the buffer to 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole,1 mM 2-mercaptoethanol, 6 M urea pH 8.0 and wash with 10 ml.

Refolding

Refolding of the bound protein is performed by the use of a linear  6--> 0 M urea gradient, starting with the wash buffer above and finishing at one without urea. A gradient volume of 30 ml or higher and a flow rate of 0.1–1 ml/min can beused, while the optimal renaturation rate should be determined experimentally for each protein. Continue to wash with 5 ml of buffer without urea after the gradient has come to its endpoint.

Elution

Elute the refolded recombinant protein using a 10–20 ml linear gradient starting with 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0. and ending with the same buffer including 500 mM imidazole.

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: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 6 M urea, 1 mM 2-mercaptoethanol pH 8.0

Refolding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0

Refolding gradient: 30 ml

Elution Buffer: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0

Elution gradient: 10 ml

Fraction volumes: 3 ml sample loading, wash and refolding, 1 ml elution

Column: Ni2+-loaded HiTrap Chelating 1 ml

Sample: N-terminal (His) 6-tagged recombinant protein produced in E.coli

Flow rates: 0.1–1 ml/min, sample loading and refolding 1 ml/min, wash and elution