Characterization of recombinant human diamine oxidase (rhDAO)produced in Chinese Hamster Ovary (CHO) cells

Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.

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 2016 Jun 10;227:120-130. doi: 10.1016/j.jbiotec.2016.04.002. Epub 2016 Apr 7.

Author information

1
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.
2
Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
3
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria.
4
Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
5
Polymun Scientific Immunbiologische Forschung GmbH, Donaustraße 99, 3400 Klosterneuburg, Austria.
6
Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
7
Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Electronic address: bernd.jilma@meduniwien.ac.at.

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.

KEYWORDS:

AOC1; Amiloride-sensitive amine oxidase; CHO; Chinese Hamster Ovary; DAO; Recombinant human diamine oxidase

PMID:
27063138
DOI:
10.1016/j.jbiotec.2016.04.002

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