Expression and characterization of a recombinant HSulf-2. (A) Domains in HSulf-2 are schematically presented. Positions of peptides that are used to develop polyclonal antibodies are shown. A recombinant protein with a N-terminal FLAG-tag (F) (rHSulf-2) and an enzymatically inactivated protein (rΔCCHSulf-2) were designed and produced as described . (B) Purified rHSulf-2 and rΔCCHSulf-2 produced in 293 cells were blotted with H2.3 anti-HSulf-2 antibody. A 72-kDa and a 74 kDa band were detected, respectively. rHSulf-2 was also blotted with a FLAG antibody. (C) Endoglucosamine-6-sulfatase activity of rHSulf-2 was measured as a function of enzyme concentration (left panel, 4 h incubation time) and reaction time (right panel, 240 ng of rHSulf-2) as described in Methods. rHSulf-2 liberated sulfate groups on C-6 position of glucosamine residues within trisulfated disaccharide units in heparin.