Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography.
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 beta-cyclodextrin (beta-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 beta-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 (0.6-0.8 mg/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.
Dong
Xiao-Yan
XY
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Chen
Li-Jun
LJ
Sun
Yan
Y
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000302:isolation & purification; D008670:Metals / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000737:chemistry
D002593:Cetrimonium Compounds; C471213:His-His-His-His-His-His; D008670:Metals; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; C031215:betadex; D000077286:Cetrimonium