RECOMBINANT SOLUBLE BETA-1,4-GALACTOSYLTRANSFERASES EXPRESSED IN SACCHAROMYCES-CEREVISIAE - PURIFICATION, CHARACTERIZATION AND COMPARISON WITH HUMAN ENZYME

beta-1,4-Galactosyltransferase (Gal-T, EC 2.4,1.38) transfers galactos e (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc- R. Soluble Gal-T, purified from human breast milk. was shown to be ver y heterogeneous by isoelectric focusing (IEF). In order to produce suf ficient homogeneous enzyme for three-dimensional analysis, the human e nzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, product ion scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The pu rification protocol was based on chromatography on concanavalin-A-Seph arose followed by affinity chromatographies on GlcNAc-Sepharose and al pha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosyl ation at the single N-glycosylation site, preventing recognition by an tibodies. 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 an d showed only three isoforms by IEF analysis. Then we abolished the si ngle N-glycosylation consensus sequence by site-directed mutagenesis c hanging Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal -T) was expressed in S. cerevisiae and its production scared 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 ba nds. The O-glycans could be removed by jack bean alpha-mannosidase tre atment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No sign ificant changes in CD spectra were observed between hGal-T and N-degly cosylated rGal-T. Light-scattering analysis revealed dimerization of b oth enzymes. These data indicate that N-deglycosylated rGal-T was corr ectly folded, homogeneous and thus suitable for crystallization experi ments.