Paul.xu at 22:10, 12 September 2019

2019-09-12T22:10:44Z

New page

== 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
[[Category:Protocols]]

On Column Refolding - Revision history

Paul.xu at 22:10, 12 September 2019