Abstract
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.