title: Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris.
abstract: The methylotrophic yeast Pichia pastoris is often used as an organism for the heterologous expression of proteins and has been used already for production of a number of glycosyltransferases involved in the biosynthesis of N- and O-linked oligosaccharides. In our recent studies, we have examined the expression in P. pastoris of Arabidopsis thaliana and Drosophila melanogaster core alpha1,3-fucosyltransferases (EC 2.4.1.214), A. thaliana beta1,2-xylosyltransferase (EC 2.4.2.38), bovine beta1,4-galactosyltransferase I (EC 2.4.1.38), D. melanogaster peptide O-xylosyltransferase (EC 2.4.2.26), D. melanogaster and Caenorhabditis elegans beta1,4-galactosyltransferase VII (SQV-3; EC 2.4.1.133) and tomato Lewis-type alpha1,4-fucosyltransferase (EC 2.4.1.65). Temperature, cell density and medium formulation have varying effects on the amount of activity resulting from expression under the control of either the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) or inducible alcohol oxidase (AOX1) promoters. In the case of the A. thaliana xylosyltransferase these effects were most pronounced, since constitutive expression at 16 degrees C resulted in 30-times more activity than inducible expression at 30 degrees C. Also, the exact nature of the constructs had an effect; whereas soluble forms of the A. thaliana xylosyltransferase and fucosyltransferase were active with N-terminal pentahistidine tags (in the former case facilitating purification of the recombinant protein to homogeneity), a C-terminally tagged form of the A. thaliana fucosyltransferase was inactive. In the case of D. melanogaster beta1,4-galactosyltransferase VII, expression with a yeast secretion signal yielded no detectable activity; however, when a full-length form of the enzyme was introduced into P. pastoris, an active secreted form of the protein was produced.
authors.affiliation: Glycobiology Division, Institut für Chemie, Universität für Bodenkultur, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Monika
authors.initials: M
authors.lastname: Bencúrová
authors.forename: Dubravko
authors.initials: D
authors.lastname: Rendić
authors.forename: Gustáv
authors.initials: G
authors.lastname: Fabini
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Kopecky
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000235:genetics; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis / Q000235:genetics; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D005647:Fucosyltransferases; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; C473070:beta4GalT7 protein, Drosophila; C031692:galactoside 3-fucosyltransferase; D009237:N-Acetyllactosamine Synthase; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase