Recombinant Soluble β‐1,4‐Galactosyltransferases Expressed in Saccharomyces cerevisiae

Abstract

β-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from JDP-Gal to N -acetyl-d-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A–Sepharose followed by affinity chromatographies on GlcNAc–Sepharose and α-lactalbumin–Sepharose. Analysis by SDS/PAGE revealed hyper-glycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69→Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U. N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean α-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGa1-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.