Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska.
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska.
Curr Protoc. 2021 Nov;1(11):e301. doi: 10.1002/cpz1.301.
Protein tyrosine O-sulfation (PTS) is a post-translational modification (PTM) that occurs exclusively on secreted and membrane-bound proteins. It participates in a wide range of important biological processes and is involved in the development of many diseases. The biomedical importance of PTS can only be fully unveiled when the right chemical/biological tools are available. This article outlines the steps for using an engineered Escherichia coli tyrosyl-tRNA synthetase to genetically encode sulfotyrosine (sTyr) for biological investigations of PTS in mammalian cells. Two basic protocols are described to demonstrate this methodology, including the site-specific incorporation of sTyr into eGFP protein in HEK293T cells and the functional study of an sTyr-containing CXCR4 protein using a calcium mobilization assay. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Site-specific incorporation of sTyr into eGFP Basic Protocol 2: Functional study of site-specifically sulfated CXCR4.
蛋白质酪氨酸 O-硫酸化 (PTS) 是一种仅发生在分泌型和膜结合蛋白上的翻译后修饰 (PTM)。它参与了广泛的重要生物学过程，并与许多疾病的发展有关。只有当有合适的化学/生物学工具时，PTS 的生物医学重要性才能被充分揭示。本文概述了使用工程化的大肠杆菌酪氨酸 tRNA 合成酶将硫酸酪氨酸 (sTyr) 基因编码到哺乳动物细胞中进行 PTS 生物学研究的步骤。描述了两种基本方案来演示该方法，包括在 HEK293T 细胞中定点掺入 sTyr 到 eGFP 蛋白中，以及使用钙动员测定法对含有 sTyr 的 CXCR4 蛋白进行功能研究。© 2021 Wiley Periodicals LLC. 基本方案 1：定点掺入 sTyr 到 eGFP 中 基本方案 2：定点硫酸化 CXCR4 的功能研究。
