BOKU - Universität für Bodenkultur Wien - Forschungsinformationssystem
Gewählte Publikation:
Gludovacz, E; Maresch, D; Bonta, M; Szollosi, H; Furtmuller, PG; Weik, R; Altmann, F; Limbeck, A; Borth, N; Jilma, B; Boehm, T.
(2016):
Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells
J BIOTECHNOL. 2016; 227: 120-130.
FullText
FullText_BOKU
- 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 TPQand 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. (C) 2016 Elsevier B.V. All rights reserved.
- Autor*innen der BOKU Wien:
- Altmann Friedrich
- Borth Nicole
- Furtmüller Paul Georg
- Gludovacz Elisabeth
- Maresch Daniel
- Find related publications in this database (Keywords)
- DAO
- Recombinant human diamine oxidase
- CHO
- Chinese Hamster Ovary
- Amiloride-sensitive amine oxidase
- AOC1
Altmetric: