Goda Natsuko, Matsuo Naoki, Tenno Takeshi, Ishino Sonoko, Ishino Yoshizumi, Fukuchi Satoshi, Ota Motonori, Hiroaki Hidekazu
Laboratory of Structural Molecular Pharmacology; Graduate School of Pharmaceutical Sciences; Nagoya University; Furocho, Chikusa-ku, Nagoya, Japan; These authors equally contributed to the work.
Laboratory of Structural Molecular Pharmacology; Graduate School of Pharmaceutical Sciences; Nagoya University; Furocho, Chikusa-ku, Nagoya, Japan; The Structural Biology Research Center and Division of Biological Science; Graduate School of Science; Nagoya University; Furocho, Nagoya, Japan.
Intrinsically Disord Proteins. 2015 Feb 23;3(1):e1011004. doi: 10.1080/21690707.2015.1011004. eCollection 2015.
Intrinsically disordered proteins (IDPs) are an emerging concept. IDPs have high flexibility in their polypeptide chains, lacking a stable 3-dimensional structure. Because of the difficulty in performing X-ray crystallography for IDPs, nuclear magnetic resonance (NMR) spectroscopy is the first choice for atomic-level investigation of their nature. Given that isotopically labeled IDP samples are necessary for NMR study, a robust and cost-effective protocol for bacterial expression and purification of IDP is also needed. We employed the N (EDDIE)-autoprotease fusion protein system. Although IDPs are believed to be readily degraded by endogenous proteases when expressed in , N-fused IDPs showed excellent resistance to degradation. Seven IDPs of uncharacterized function sampled from the human genome as well as 3 constructs from IDP regions derived from human FancM and Hef were prepared. We improved the protocol of refolding of N (EDDIE) to use dialysis, which is convenient for subsequent purification using reversed-phase (RP) HPLC. The method is robust and widely applicable to any IDP sample, promoting the acquisition of experimental data for IDPs in a high-throughput manner.
内在无序蛋白质（IDP）是一个新兴概念。IDP的多肽链具有高度灵活性，缺乏稳定的三维结构。由于对IDP进行X射线晶体学分析存在困难，核磁共振（NMR）光谱法是对其性质进行原子水平研究的首选方法。鉴于NMR研究需要同位素标记的IDP样品，因此还需要一种用于细菌表达和纯化IDP的强大且经济高效的方案。我们采用了N（EDDIE）-自蛋白酶融合蛋白系统。尽管人们认为IDP在细菌中表达时容易被内源性蛋白酶降解，但N融合的IDP表现出优异的抗降解能力。我们制备了从人类基因组中选取的7种功能未知的IDP，以及来自人类FancM和Hef的IDP区域的3种构建体。我们改进了N（EDDIE）的重折叠方案，采用透析法，这便于随后使用反相（RP）HPLC进行纯化。该方法强大且广泛适用于任何IDP样品，以高通量方式促进了IDP实验数据的获取。
