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.

中文翻译：

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用于蛋白质组蛋白处理的INSOL标签的开发及其在蛋白质阵列分析中的应用。

蛋白质组学分析需要能够实现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，该标签衍生自人类转录因子MafG。带有INSOL标签的靶蛋白在真核体外表达系统中表达，并在19,000×g离心20分钟后以沉淀形式回收。使用111种胞质蛋白对GST标签和INSOL标签的目标蛋白回收率进行比较后，发现INSOL标签蛋白的产量提高了四倍。随后，我们使用使用INSOL-tag纯化的267种癌症抗原，开发了一种INSOL-CTA阵列方法，用于分析癌症患者血清中的自身抗体。阵列的检测下限约为11.1 pg IgG，并且与ELISA的相关性很高（R2 = .993，.955）。此外，当对消化道癌症患者的血清进行自身抗体谱分析时，观察到抗原扩散。这些数据表明，INSOL标签是一种通用标签，可以使多种靶蛋白不溶。因此，它有望成为用于蛋白质阵列，抗体生产和质谱的综合蛋白质制备的强大工具。