title: Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.
abstract: Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria; Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Gludovacz
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Bonta
authors.affiliation: Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
authors.forename: Helen
authors.initials: H
authors.lastname: Szöllösi
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Furtmüller
authors.affiliation: Polymun Scientific Immunbiologische Forschung GmbH, Donaustraße 99, 3400 Klosterneuburg, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Weik
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Limbeck
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Nicole
authors.initials: N
authors.lastname: Borth
authors.affiliation: Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Electronic address: bernd.jilma@meduniwien.ac.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Jilma
authors.affiliation: Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Boehm
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003067:Coenzymes / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010659:Phenylhydrazines / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D008670:Metals; D010455:Peptides; D010659:Phenylhydrazines; D011134:Polysaccharides; D011994:Recombinant Proteins; C030299:phenylhydrazine; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D006631:Amine Oxidase (Copper-Containing)
keywords: AOC1; Amiloride-sensitive amine oxidase; CHO; Chinese Hamster Ovary; DAO; Recombinant human diamine oxidase