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E Gludovacz, D. Maresch, M. Bonta, H Szöllösi, P Furtmüller, R Weik, F. Altmann, A. Limbeck, N Borth, B Jilma, T Boehm:
"Characterization of recombinant human diamine oxidase (rhDAO)produced in Chinese Hamster Ovary (CHO) cells";
Journal of Biotechnology, 227 (2016), 120 - 130.

tHuman diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activ-ities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAOhas been characterized after isolation from either native sources or the heterologous production in insectcells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patternsthat may alter its biochemical properties.We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three steppurification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites wereoccupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titra-tion. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the activesite was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity ofthe protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra datawere used to characterize structural properties. The substrate preference and kinetic parameters werein accordance with previous publications.The establishment of a recombinant production system for hDAO enables us to generate decentamounts of protein with negligible impurities to address new scientific questions.

Recombinant human diamine oxidase, Chinese Hamster Ovary, Amiloride-sensitive amine oxidase, AOC1a

http://dx.doi.org/10.1016/j.jbiotec.2016.04.002