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 beta-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 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T 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.
Krezdorn
C H
CH
Physiologisches Institut, Universität Zürich, Switzerland.
Watzele
G
G
Kleene
R B
RB
Ivanov
S X
SX
Berger
E G
EG
D006031:Glycosylation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase