title: Role of urea on recombinant Apo A-I stability and its utilization in anion exchange chromatography.
abstract: Apolipoprotein A-I (Apo A-I) is an important lipid-binding protein involved in the transport and metabolism of cholesterol. High protein purity, in particular with respect to endotoxins is required for therapeutic applications. The use of urea during the purification process of recombinant Apo A-I produced in Escherichia coli has been suggested so as to provide high endotoxin clearance. In this work, we show that urea can be used as a sole modifier during the ion exchange chromatographic purification of Apo A-I and we investigate the molecular mechanism of elution by correlating the effect of urea on self-association, conformation and adsorption equilibrium properties of a modified model Apo A-I. In the absence of urea the protein was found to be present as a population of oligomers represented mainly by trimers, hexamers and nonamers. The addition of urea induced oligomer dissociation and protein structure unfolding. We correlated the changes in protein association and conformation with variations of the adsorption equilibrium of the protein on a strong anion exchanger. It was confirmed that the adsorption isotherms, described by a Langmuir model, were dependent on both protein and urea concentrations. Monomers, observed at low urea concentration (0.5M), were characterized by larger binding affinity and adsorption capacity compared to both protein oligomers (0M) and unfolded monomers (2-8M). The reduction of both the binding strength and maximum adsorption capacity at urea concentrations larger than 0.5M explains the ability of urea of inducing elution of the protein from the ion exchange resin. The dissociation of the protein complexes occurring during the elution could likely be the origin of the effective clearance of endotoxins originally trapped inside the oligomers.
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland.
authors.forename: Monica
authors.initials: M
authors.lastname: Angarita
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland.
authors.forename: Paolo
authors.initials: P
authors.lastname: Arosio
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland; ChromaCon AG, Zurich, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Müller-Späth
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Baur
authors.affiliation: Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Roberto
authors.initials: R
authors.lastname: Falkenstein
authors.affiliation: Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kuhne
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland. Electronic address: massimo.morbidelli@chem.ethz.ch.
authors.forename: Massimo
authors.initials: M
authors.lastname: Morbidelli
mesh_terms: D000327:Adsorption; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange / Q000379:methods*; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014508:Urea / Q000737:chemistry*
chemical_list: D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D014508:Urea
keywords: Apolipoprotein; Denaturation; Ion exchange equilibrium; Protein adsorption; Self-association; Urea