Tallarita Elena, Pollegioni Loredano, Servi Stefano, Molla Gianluca
Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, Varese, Italy.
Protein Expr Purif. 2012 Apr;82(2):345-51. doi: 10.1016/j.pep.2012.01.021. Epub 2012 Feb 8.
The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ(1)-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure-function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.
人类脯氨酸氧化还原酶（PRODH）基因已被证明在调节细胞存活和凋亡途径中具有独特作用，并且与心脏颜面综合征/迪格奥尔格综合征以及精神分裂症易感性增加有关。它编码黄素蛋白脯氨酸氧化酶（PO），该酶催化L-脯氨酸转化为Δ(1)-吡咯啉-5-羧酸。尽管人类PO在生理和医学方面具有重要意义，但到目前为止，只有PO的微生物同源物被表达为重组蛋白并得到充分表征。通过生物信息学分析来确定催化结构域和具有高度内在结构紊乱倾向的区域，我们设计了人类PO的缺失变体，这些变体在大肠杆菌中成功表达为可溶性蛋白，产量相当高（高达10mg/L发酵液）。His标签的PO-barrelN蛋白被分离为一种活性（比活性为0.032U/mg蛋白）的二聚体全酶，显示出含FAD的黄素蛋白氧化酶的典型光谱特性。这些结果为阐明这种人类黄素酶的结构-功能关系以及阐明与疾病状态相关的报道多态性的影响铺平了道路。
