Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari "A. Moro", Bari, Italy.
Methods Mol Biol. 2021;2280:69-85. doi: 10.1007/978-1-0716-1286-6_6.
Here we describe a protocol for a one-step purification of a soluble form of human FAD synthase (isoform 2; hFADS2), overexpressed as a 6-His-tagged fusion protein in Escherichia coli, with a yield of about 15 mg from 1 L of transformed bacterial culture.Following a desalting procedure, the protein is obtained in its FAD-bound form (about 0.8 molecules of FAD per 1 protein monomer). A simple method is also proposed here, for the rapid estimation of the [FAD ]/[protein monomer] ratio, starting from the typical flavoprotein spectrum of the purified protein fraction.The procedure described gives the protein at a quite high grade of purity (about 95%) and in its bifunctional (2.7.7.2/3.6.1.18) enzymatically active form, useful for further kinetical and molecular characterization.
在这里，我们描述了一种一步法纯化可溶性人 FAD 合酶（同种型 2；hFADS2）的方案，该酶以 6-His 标记的融合蛋白形式在大肠杆菌中过表达，从 1 升转化细菌培养物中约可获得 15 毫克。经过脱盐处理后，该蛋白以其结合 FAD 的形式获得（每个蛋白单体约有 0.8 个 FAD 分子）。本文还提出了一种简单的方法，可从纯化蛋白部分的典型黄素蛋白光谱出发，快速估算 [FAD]/[蛋白单体]比值。所描述的方案可提供相当高纯度的蛋白（约 95%），并保持其双功能（2.7.7.2/3.6.1.18）酶活性形式，可用于进一步的动力学和分子特征分析。
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