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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 …