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首页> 外文期刊>Biochemistry >Screening of Selective Inhibitors of 1 alpha,25-Dihydroxyvitamin D-3 24-Hydroxylase Using Recombinant Human Enzyme Expressed in Escherichia coli
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Screening of Selective Inhibitors of 1 alpha,25-Dihydroxyvitamin D-3 24-Hydroxylase Using Recombinant Human Enzyme Expressed in Escherichia coli

机译：使用在大肠杆菌中表达的重组人酶筛选1α，25-二羟基维生素D-3 24-羟化酶的选择性抑制剂

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摘要

High-level heterologous expression of human 1 alpha,25-dihydroxyvitamin D-3 24-hydroxylase (CYP24A1) in Escherichia coli was attained via a fusion construct by appending the mature CYP24A1 without the leader sequence to the maltose binding protein (MBP). Facile purification was achieved efficiently through affinity chromatography and afforded fully functional enzyme of near homogeneity, with a k(cat) of 0.12 min(-1) K-M of 0.19 mu M toward 1 alpha,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3]. A convenient and reliable cell-free assay was established and used to screen vitamin D analogues with potential inhibitory properties toward CYP24A1. Some of the compounds exhibited potent inhibition with K-1 values as low as 0.021 mu M. Furthermore, TS17 and CPA1 exhibited superior specificity toward CYP24A1 over 25-hydroxyitamin D-3 1 alpha-hydroxylase (CYP27B1), with selectivities of 39 and 80, respectively. Addition of TS17 or CPA1 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D-3 in the medium. Their activities in vitamin D receptor (VDR) binding, CYP24A1 transcription, and HL-60 cell differentiation were evaluated as well.
机译：通过将没有前导序列的成熟CYP24A1附加到麦芽糖结合蛋白（MBP）上，通过融合构建体获得人1α，25-二羟基维生素D-3 24-羟化酶（CYP24A1）在大肠杆菌中的高水平异源表达。通过亲和色谱有效地实现了简便纯化，并提供了接近均一的全功能酶，对1个alpha，25-dihydroxyvitamin D-3 [1,25（OH）的ak（cat）为0.12 min（-1）KM，为0.19μM。 ）（2）D-3]。建立了方便，可靠的无细胞检测方法，并将其用于筛选对CYP24A1具有潜在抑制作用的维生素D类似物。一些化合物表现出有效的抑制作用，其K-1值低至0.021μM。此外，TS17和CPA1对CYP24A1的特异性优于25-羟基维生素D-3 1α-羟化酶（CYP27B1），选择性分别为39和80 ， 分别。在小鼠成骨细胞培养物中添加TS17或CPA1可以维持培养基中1,25（OH）（2）D-3的水平。还评估了它们在维生素D受体（VDR）结合，CYP24A1转录和HL-60细胞分化中的活性。

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