Heparan sulfate proteoglycans (HSPGs) play important roles in human physiol., including in cell signaling, blood coagulation, and the immune response.The biosynthesis of heparan sulfate (HS), the carbohydrate component of HSPGs, takes place in the endoplasmic reticulum and the Golgi apparatusIn addition to the action of glycosyl transferase and epimerase enzymes in this process, a family of Golgi-resident, membrane-anchored sulfotransferases acts to confer specific sulfation patterns on HS, which have important implications for its function.At Tango Therapeutics, one of these enzymes, heparan sulfate-2-O-sulfotransferase (HS2ST1), was identified as a potential target of interest for drug discovery.In order to enable drug discovery efforts, we report here the large-scale expression and purification of the soluble luminal domain of human HS2ST1 from mammalian cells, measurement of its activity in a coupled assay format suitable for high-throughput screening, and the first crystal structures of human HS2ST1 in the apo and PAP-bound forms.Moreover, we used our assay to characterize a small set of active site mutants previously reported to have diminished activity.Collectively, these results establish the feasibility of conducting large-scale hit finding campaigns to identify inhibitors of this novel target.