Expression in Escherichia coli, purification and kinetic characterization of human heparan sulfate 3-O-sulfotransferase-1.

Heparan sulfate (HS) glycosaminoglycans are a structurally diverse class of complex biomolecules that modulate many important events at the cell surface and within the extracellular matrix and whose structural heterogeneity derives largely from the sequence-specific N- and O-sulfations catalyzed by an extensive repertoire of sulfating enzymes. We have expressed the human heparan sulfate 3-OST-1 isoform in Escherichia coli and subsequently purified a soluble, active enzyme. To assess its functionality, we determined the kinetic parameters for the recombinant 3-O-sulfotransferase-1 using a radiochemical assay that directly measures the 3-O-sulfation of unlabeled bovine kidney heparan sulfate in vitro using [(35)S]PAPS as the sulfate donor. The apparent K(m) values measured were in the low micromolar range (K(HS)(m) = 4.3 microM; K(PAPS)(m) = 38.6 microM); V(max) values of 18 and 21 pmol sulfate/min/pmol of enzyme for HS and PAPS, respectively. These values were compared with kinetic parameters likewise measured for recombinant 3-OST-1 purified from baculovirus-infected sf9 cells. The two enzymes appear to modify heparan sulfate in vitro to roughly the same extent and with comparable specificities. The expression of 3-OST-1 in E. coli represents an important step in subsequent structure-function studies. ©2002 Elsevier Science (USA).