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Application note 18-1134-37 AC
Affinity chromatography
Rapid and efficient purification and refolding of a (histidine)6-tagged recombinant protein produced in E. coli as inclusion bodies
Summary
This Application Note describes the purification and
refolding of a recombinant protein tagged with a (histidine)6-tag at its N-terminus. Using a simple but efficient purification and refolding procedure, a protein initially produced as intracellular inclusion body material in Escherichia coli is converted to soluble protein exhibiting the desired activity.This protocol has been ud successfully for veral different (histidine)6-tagged recombinant proteins.
Introduction
Heterologous expression of foreign genes in E. coli can be engineered to lead to either intracellular accumulation of recombinant protein, or to cretion and accumulation in the periplasmic space. While the latter mode of expression is sometimes advantageous in terms of protein folding, solubility, and cysteine oxidation, the magnitude of protein production is generally much higher when intracellular expression is ud (1).
However, recombinant protein accumulated intracellularly is frequently laid down in the form of inclusion bodies,
insoluble aggregates of misfolded protein lacking biological activity (2,3,4,5). The high buoyant density of inclusion bodies facilitates their paration from soluble E. coli
proteins and cell debris by differential centrifugation (4,6,7). Conventional methods for refolding of insoluble recombinant proteins include slow dialysis or dilution into a buffer of near-neutral pH (8). Gel filtration, ion exchange, or
hydrophobic interaction chromatography have been ud (9,10,11) to facilitate the refolding step.
Affinity tagging of the recombinant protein, for example by the addition of veral concutive histidine residues,
makes the efficient purification and refolding in a single
chromatographic step possible. Since binding of the histidine tract to immobilized divalent metal ions can occur in the prence of a chaotropic agent (such as urea or guanidine hydrochloride) at high concentration, (histidine)6-tagged inclusion body protein can be solubilized by chaotropic extraction and directly bound to an affinity matrix. Removal of contaminating proteins and refolding by exchange to non-denaturing buffer conditions can then be performed before elution of the protein from the column (12).
A general protocol for the purification and refolding of a (histidine)6-tagged recombinant protein produced in E. coli is shown in Figure 1.
Fig 1. General scheme for the extraction, solubilization, and refolding of (histidine)6-tagged recombinant proteins produced as inclusion bodies in Escherichia coli cells.
Disruption, wash, and isolation of inclusion bodies
Resuspend the cell paste from a 100 ml culture of E. coli
expressing (histidine)
6-tagged recombinant protein in 4 ml
20 mM Tris-HCl pH 8.0. Disrupt the cells with sonication
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on ice (e.g., 4 × 10 c.) 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 M NaCl, 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 cond round of urea wash. At this stage the pellet material can be washed once in buffer lacking urea, and then st
ored 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.
Proceed directly with the purification and refolding steps. Preparation of the column
HiTrap™ Chelating HP 1 ml column is washed with 5 ml distilled water using a 5 ml syringe. Load 0.5 ml 0.1 M
NiSO
4 and continue to wash with
5 ml distilled water.
Equilibrate the column with 5–10 ml 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-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-mercapto-ethanol 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 using 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 be ud, 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,
仙神河大桥20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0 and ending with the same buffer including 500 mM imidazole (Fig 2).
Fractions containing the eluted protein are pooled and subjected to buffer exchange using a HiTrap Desalting or PD-10 column, in order to remove imidazole. The refolded (histidine)
6
-tagged pr ot ein is now ready for analysis of biological activity.
The choice of HiTrap column size depends on the amount of expresd protein.
While in this example a HiTrap Chelating HP 1 ml column
is ud, a HiTrap Chelating HP 5 ml is also available and should be ud if the expected amount of recombinant protein exceeds 10 mg. For further scaling-up, Chelating Sepharo™ Fast Flow is available.
1.0
0.75
0.5
0.25
A280
Fig 2. On-column refolding and purification of a (histidine)
6
-tagged protein from inclusion bodies on Ni2+-charged HiTrap Chelating HP.
Column: Ni2+-loaded HiTrap Chelating HP 1 ml
Sample:N-terminal (histidine)
6
-tagged recombinant protein produced
li
Flow rates:0.1–1 ml/min, sample loading and refolding 1 ml/min, wash
and elution
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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
E lution gradient: 10 ml
Fraction volumes: 3 ml sample loading, wash and refolding 1 ml elution
2 Application note 18-1134-37 AC
Application note 18-1134-37 AC 3
Analysis
The aggregation state and purity of the refolded
(histidine)6-tagged recombinant protein eluted from HiTrap Chelating HP is checked by gelfiltration on Superdex 75 HR 10/30 (Figure 3) and SDS-PAGE (Figure 4).
Regeneration and storage
肝气横逆Regenerate the column with 5 ml 6 M guanidine
hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 50 mM EDTA, pH 8.0. Wash with 10 ml distilled water followed by 10 ml 20% ethanol. Store the column in 20% ethanol.
References:
1. Marston, F.A.O. The purification of eucaryotic polypeptides synthesized in Escherichia coli. Bioc
hem J. 240, pp 1–12 (1986).
2. Williams, D.C., Van Frank, R.M., Muth, W.L., Burnett, J.P. Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science 215, pp. 687–689 (1982).
3. Harris, T.J.R. Expression of eucaryotic genes li. In: Williamson, R.(Ed.) Genetic Engineering . Vol. 4, Academic Press, London, pp. 127–185 (1983).
4. Marston, F.A.O., Lowe, P.A., Doel, M., Schoemaker, J.M., White, S., Angal, S. Purification of calf prochymosin (prorennin) synthesized in Escherichia coli. Bio/Technology 2, 800–804 (1984).
5. Lowe, P.E., et al. Solubilization, refolding and purification of eucaryotic proteins expresd li, in: Protein purification: Micro to Macro , A.R. Liss, Inc., pp. 429-442 (1987).
6. Kelley, R.F., Winkler, M.E. Folding of eucaryotic proteins produced in Escherichia coli. Genetic Engineering 12, pp. 1–19 (1990).
7. Mitraki, A., King, J. Protein folding intermediates and inclusion body formation. Bio/Technology 7, pp. 690–697 (1990).
8. Knuth, M.W., Burgess, R.R. Purification of proteins in the denaturated state, in: Protein purification: Micro to Macro , A. R. Liss, Inc., pp. 279–305 (1987).
9. Werner, M.H., Clore, G.M., Gronenborn, A.M., Kondoh, A., Fisher, R.J. Refolding proteins by gelfiltration chromatography. FEBS Letter 345, pp. 125–130 (1994).10. Hoess, A., Arthur, A.K., Wanner, G., Fanning, E. Recovery of soluble, biologically active recombinant proteins from total bacterial lysates using ion exchange resin. Bio/Technology 6, pp. 1214–1217 (1988).
11. Application Note: Purification and renaturation of recombinant proteins produced in Escherichia coli as inclusion bodies. GE Healthcare 18-1112-33.12. Colangeli, R., Heijbel, A., Williams, A.M., Manca, C., Chan, J., Lyashchenko, K., Gennaro, M.L. Three-step purification of lipopolysaccharide-free polyhistidine- tagged recombinant antigens of Myobacterium tuberculosis. J of Chromatography B, 714, pp. 223–235 (1998).
Gel: PhastGel™ Gradient 10–15
Sample Dilution 1:5 with 15% SDS, 30% 2-mercaptoethanol, pretreatment: 10 mM Tris, 1 mM EDTA Sample volume : 1 µl
Molecular weight standard : Low Molecular Markers
Staining: Coomassie TM , according to the manufacturer’s standard
protocol
Instrument:
PhastSystem™
1 2 3 4 5 6 7 8Lane 1: Low Molecular Markers
Lane 2: Starting material for HiTrap Chelating 1 ml Lane 3: Fraction 1 Gua-HCl wash (manually)Lane 4: Fraction 2 Gua-HCl wash (manually)Lane 5: Fraction 3 Gua-HCl wash (manually)Lane 6:
Fraction 4 Gua-HCl
wash (manually) Lane 7: Fraction 1 Urea wash (manually)Lane 8: Fraction 2 Urea
wash (manually)M r 9700066000450003000020100
14400
Fig 4. SDS-PAGE analysis.
1.0
0.75
0.5
0.25
5
10
15
20
服务工作总结ml
A 280
Fig 3. Analysis using gel filtration of refolded (histidine)6-tagged protein.
Column: Superdex TM 75 HR 10/30 (V T : 24 ml)
Sample: 0.2 ml purified and refolded N-terminal (histidine)6-tagged
日记50字左右recombinant protein eluted from HiTrap Chelating HP 1 ml Buffer: 0.15 M NaCl Flow rate:
0.5 ml/min Fraction volume:
1 ml
M r 970006600045000300002010014400
Lane 1: Low Molecular Markers Lane 2: Fraction 38
Lane 3: Fraction 39 Lane 4: Fraction 40 Lane 5: Fraction 41 Lane 6: Fraction 42
Lane 7: Fraction 46 Lane 8:
Fraction 49
1 2 3 4 5 6 7 8
GE, imagination at work, and GE monogram are trademarks of General Electric Company.
ÄKTAdesign, Drop Design, HiLoad, HiPrep, HiTrap, PhastGel, PhastSystem, Sepharo, and Superdex, are trademarks of GE Healthcare companies.
All third party trademarks are the property of their respective owners.
Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a licen under US patent numbers 5,284,933 and 5,310,663 and equivalent patents and patent applications in other countries (assignee: Hoffman La Roche, Inc).
© 1999-2007 General Electric Company – All rights rerved. First published Sep. 1999.
All goods and rvices are sold subject to the terms and
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18-1134-37 AC 04/2007
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Ordering information
Product
Quantity
Code No.
HiTrap Chelating HP 5 × 1 ml 17-0408-01HiTrap Chelating HP 1 × 5 ml 17-0409-01HiTrap Chelating HP
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5 × 5 ml
17-0409-03
Related literature
Code No.
Recombinant Protein Purification Handbook, Principles and methods 18-1142-75Affinity Chromatography,
Columns and Media Selection Guide 18-1121-86Affinity Chromatography
Handbook, Principles and Methods 18-1022-29HiTrap Column Guide
18-1129-81
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