Expression of functional soluble forms of human beta-1, 4-galactosyltransferase I, alpha-2,6-sialyltransferase, and alpha-1, 3-fucosyltransferase VI in the methylotrophic yeast Pichia pastoris.
The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides.
Malissard
M
M
Institute of Physiology, University of Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland. malissar@physiol.unizh.ch
Zeng
S
S
Berger
E G
EG
D003001:Cloning, Molecular / Q000379:methods; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007788:Lactose Synthase / Q000235:genetics* / Q000378:metabolism*; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics / Q000502:physiology; D010843:Pichia; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000072235:Mating Factor; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D007788:Lactose Synthase; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase