title: Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker's yeast using an expanded bed adsorption system.
abstract: Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers' yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that charging with Zn2+ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers' yeast diluted to 10 mg/ml total protein content with a recovery of 80-100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.
authors.affiliation: Department of Biochemical Engineering, University College London, UK.
authors.forename: N A
authors.initials: NA
authors.lastname: Willoughby
authors.forename: T
authors.initials: T
authors.lastname: Kirschner
authors.forename: M P
authors.initials: MP
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Hjorth
authors.forename: N J
authors.initials: NJ
authors.lastname: Titchener-Hooker
mesh_terms: D000327:Adsorption; D000426:Alcohol Dehydrogenase / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004492:Edetic Acid; D006639:Histidine / Q000032:analysis; D007093:Imidazoles / Q000032:analysis; D008670:Metals / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007093:Imidazoles; D008670:Metals; D012997:Solvents; D006639:Histidine; D004492:Edetic Acid; D000426:Alcohol Dehydrogenase