PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN BETA-1-4 GALACTOSYLTRANSFERASE EXPRESSED IN SACCHAROMYCES-CEREVISIAE

A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell,8-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum. Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by H-1-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T. The recombinant galT was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly. \ This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.