Development of INSOL‐tag for proteome‐wide protein handling and its application in protein array analysis
Development of INSOL‐tag for proteome‐wide protein handling and its application in protein array analysis
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用于全蛋白质组蛋白质处理的 INSOL 标签的开发及其在蛋白质阵列分析中的应用
DOI：
10.1111/gtc.12735
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发表时间：
2019
期刊：
影响因子：
2.1
通讯作者：
Goshima Naoki
中科院分区：
文献类型：
--
作者：
Fukuda Eriko;Mori Masatoshi;Shiku Hiroshi;Miyahara Yoshihiro;Kawamura Yoshifumi;Ogawa Koji;Ogura Toshihiko;Goshima Naoki
蛋白质组学分析需要能够进行高通量处理的蛋白质标签;然而，目前缺乏可用于体外表达系统的通用标签。在这项研究中，我们开发了一种不溶性蛋白质标签，INSOL‐tag，来自人类转录因子MafG。INSOL标记的靶蛋白在真核体外表达系统中表达，并在19，000 × g离心20 min后以沉淀物形式回收。使用111种细胞质蛋白比较GST-tag和INSOL-tag的靶蛋白回收率，发现INSOL-tag蛋白的产量增加了4倍。使用267种用INSOL标签纯化的癌抗原，我们随后开发了INSOL CTA阵列方法，用于分析癌症患者血清中的自身抗体。该阵列的检测限约为11.1 pg IgG，与ELISA的相关性较高（R2= 0.993，0.955）。此外，当进行消化道癌患者血清的自身抗体谱分析时，观察到抗原扩散。这些数据表明，INSOL‐tag是一种多功能标签，可以使广泛的靶蛋白不溶。因此，它有望成为一个强大的工具，在全面的蛋白质制备的蛋白质阵列，抗体生产，和质谱。
Proteomic analysis requires protein tags that enable high‐throughput handling; however, versatile tags that can be used in in vitro expression systems are currently lacking. In this study, we developed an insoluble protein tag, INSOL‐tag, derived from human transcription factor MafG. The INSOL‐tagged target protein is expressed in a eukaryotic in vitro expression system and recovered as a pellet following centrifugation at 19,000 × g for 20 min. Comparisons of the target protein recovery rates of GST‐tag and INSOL‐tag using 111 cytoplasmic proteins revealed a fourfold increase in the yield of INSOL‐tagged proteins. Using 267 cancer antigens purified with INSOL‐tag, we subsequently developed an INSOL‐CTA array method, for profiling autoantibodies in sera of cancer patients. The detection limit of the array was approximately 11.1 pg IgG, and the correlation with ELISA was high (R2= .993, .955). Moreover, when autoantibody profiling of digestive cancer patient sera was performed, antigen spreading was observed. These data suggest that INSOL‐tag is a versatile tag that can insolubilize a wide range of target proteins. It is therefore expected to become a powerful tool in comprehensive protein preparation for protein arrays, antibody production, and mass spectrometry.