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Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

Author:
Dong, Xiao-Yan, Chen, Li-Jun, Sun, Yan
Source:
Journal of chromatography 2009 v.1216 no.27 pp. 5207-5213
ISSN:
0021-9673
Subject:
Escherichia coli, affinity chromatography, beta-cyclodextrin, cetyltrimethylammonium bromide, fluorescence, green fluorescent protein, histidine, inclusion bodies, protein folding, washing
Abstract:
This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein-CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6-0.8mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.
Agid:
280131