title: Controlled oxidative protein refolding using an ion-exchange column.
abstract: Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55% after 40 h incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling.
authors.affiliation: Centre for Biomolecular Engineering, University of Queensland, St Lucia, Qld 4072, Australia.
authors.forename: Marc
authors.initials: M
authors.lastname: Langenhof
authors.forename: Susanna S J
authors.initials: SS
authors.lastname: Leong
authors.forename: Leonard K
authors.initials: LK
authors.lastname: Pattenden
authors.forename: Anton P J
authors.initials: AP
authors.lastname: Middelberg
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D002942:Circular Dichroism; D007474:Ion Exchange; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D012710:Serum Albumin, Bovine / Q000737:chemistry*
chemical_list: D012710:Serum Albumin, Bovine