Gludovacz Elisabeth, Maresch Daniel, Bonta Maximilian, Szöllösi Helen, Furtmüller Paul G, Weik Robert, Altmann Friedrich, Limbeck Andreas, Borth Nicole, Jilma Bernd, Boehm Thomas
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.
Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
J Biotechnol. 2016 Jun 10;227:120-130. doi: 10.1016/j.jbiotec.2016.04.002. Epub 2016 Apr 7.
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.
人二胺氧化酶（hDAO）能有效降解多胺和组胺。酶活性降低可能会在孕期引发并发症，并与组胺不耐受有关。到目前为止，hDAO是在从天然来源分离或在昆虫细胞中进行异源生产后进行表征的。获取人源酶受到限制，且昆虫细胞产生的非人类糖基化模式可能会改变其生化特性。我们展示了hDAO在中国仓鼠卵巢（CHO）细胞中的异源表达以及三步纯化方案。使用电感耦合等离子体质谱（ICP-MS）分析金属含量，结果显示93%的活性位点被铜占据。使用苯肼滴定法测定了Topaquinone（TPQ）辅因子含量。94%的DAO分子含有TPQ，因此间接证实了活性位点的铜含量。进行了质谱分析以验证蛋白质的序列完整性并评估糖基化谱。利用电子圆二色性和紫外可见光谱数据来表征结构特性。底物偏好和动力学参数与先前的出版物一致。hDAO重组生产系统的建立使我们能够生成大量杂质可忽略不计的蛋白质，以解决新的科学问题。
