Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris.
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.
Bencúrová
Monika
M
Glycobiology Division, Institut für Chemie, Universität für Bodenkultur, Muthgasse 18, A-1190 Vienna, Austria.
Rendić
Dubravko
D
Fabini
Gustáv
G
Kopecky
Eva-Maria
EM
Altmann
Friedrich
F
Wilson
Iain B H
IB
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
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