title: Immobilized metal-ion affinity chromatography of recombinant Fab protein OPG C11 in the presence of EDTA-Mg(II).
abstract: Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA-Mg2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA-Mg2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA-Mg2+ treatment may have general application potentials.
authors.affiliation: xiang_hui@allergan.com
authors.forename: Hui
authors.initials: H
authors.lastname: Xiang
authors.forename: Richard
authors.initials: R
authors.lastname: Wynn
authors.forename: Lien-Hanh T
authors.initials: LH
authors.lastname: Nguyen
authors.forename: O Harold
authors.initials: OH
authors.lastname: Ross
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Ahrens
authors.forename: Karyn T
authors.initials: KT
authors.lastname: O'Neil
authors.forename: Gregory F
authors.initials: GF
authors.lastname: Hollis
authors.forename: Denis R
authors.initials: DR
authors.lastname: Patrick
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004492:Edetic Acid / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D008274:Magnesium / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D004492:Edetic Acid; D008274:Magnesium