Purification and characterization of a soluble form of the recombinant human galactose-β1,3-glucuronosyltransferase I expressed in the yeast Pichia pastoris

The galactose-β1,3-glucuronosyltransferase I (GlcAT-I) catalyzes the transfer of glucuronic acid from UDP-α-d-glucuronic acid onto the terminal galactose of the trisaccharide glycosaminoglycan–protein linker region of proteoglycans. This enzyme plays a key role in the process of proteoglycan assembly since the completion of the linkage region is essential for the conversion of a core protein into a functional proteoglycan. To investigate the enzymatic properties of human GlcAT-I, we established an expression system for producing a soluble form of enzyme in the methylotrophic yeast Pichia pastoris and developed a three-step purification procedure using a combination of anion exchange, cation exchange and heparin chromatographies. This procedure yielded 1.6 mg homogeneous enzyme from 200 ml yeast cell culture, with a specific activity value of 1.5 μmol/min/mg protein. Analysis of the specificity of GlcAT-I towards Galβ1–3Gal and Galβ1–4GlcNAc derivatives known as substrates of the β1,3-glucuronosyltransferases, showed that the enzyme exhibited a strict selectivity towards Galβ1–3Gal structures. Thus, the large source of purified active enzyme allowed the determination of the kinetic parameters of GlcAT-I towards the donor substrate UDP-GlcA and the acceptor substrate digalactoside Galβ1–3Gal.