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  • 1
    ISSN: 1573-4986
    Keywords: sialyl Lewis x ; enzymic synthesis ; N-acetylglucosaminyltransferase ; fucosyltransferase ; galactosyltransferase ; sialyltransferase ; recombinant glycosyltransferases ; P-selectin ; PSGL-1 ; β3-GalT, UDP-Gal:GalNAcα-R β-1,3-galactosyltransferase, β3-galactosyltransferase ; C2GnT, UDP-GlcNAc:Gal(β1-3)GalNAc(α1-R (GlcNAc to GalNAc) β-1,6-N-acetylglucosaminyltransferase, β6-N-acetylglucosaminyltransferase ; β4-GalT1, UDP-Gal:GlcNAc β-1,4-galactosyltransferase 1, β4-galactosyltransferase 1 ; ST3Gal3, CMP-Neu5Ac:Gal(β1-4)GlcNAc α-2,3-sialyltransferase 3, α3-sialyltransferase 3 ; α3-FucT6, GDP-Fuc:Gal(β1-4)GlcNAc (Fuc to GlcNAc) α-1,3-fucosyltransferase 6, α3-fucosyltransferase 6 ; Caco, sodium cacodylate (Na(CH3)2AsO2) - HCl buffer ; CIAP, Calf Intestinal Alkaline Phosphatase ; core 1, Gal(β1-3)GalNAc ; core 2, GlcNAc(β1-6)[Gal(β1-3)]GalNAc ; core 4, GlcNAc(β1-6)[GlcNAc(β1-3)]GalNAc ; core 6, GlcNAc(β1-6)GalNAc ; FPLC, Fast Protein Liquid Chromatography ; HPLC, High Performance Liquid Chromatography ; LacNAc, Gal(β1-4)GlcNAc ; lacto-N-biose, Gal(β1-3)GlcNAc ; MALDI TOF, Matrix Assisted Laser Desorption Ionisation Time of Flight ; MES, 2-(N-Morpholino)ethanesulfonic acid - NaOH buffer ; Me2SO, Dimethyl sulfoxide ; MLEV, Malcolm Levitt ; NMR, Nuclear Magnetic Resonance ; pNp, p-Nitrophenyl ; PSGL-1, P-Selectin Glycoprotein Ligand 1 ; ROESY, Rotating Frame Nuclear Overhauser Enhancement Spectroscopy ; sLex, Sialyl Lewis x ; TOCSY, Total Correlation Spectroscopy ; WEFT, Water Eliminated Fourier Transform
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Sialyl Lewis x (sLex) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-α-D-galactosaminide (GalNAc(α1-pNp as core substrate, the sLex-oligosaccharide Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(β1-3)GalNAc(α1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of β3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 β6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp in a yield of 〉85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk β4-galactosyltransferase 1 (EC 2.4.1.38) (yield of 〉85%), then sialylated using CMP-Neu5Ac and purified recombinant α3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human α3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 µmol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.
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  • 2
    ISSN: 1573-4986
    Keywords: saccharomyces cerevisiae ; Pichia pastoris ; β1,4galactosyltransferase ; α2,6sialytransferase ; α2,3sialytransferase ; α1,3fucosyltransferase ; α1,2mannosyltransferase ; glycosylation engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur, P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include β1,4galactosyltransferase, α2,6sialyltransferase, α2,3sialyltransferase, α1,3fucosyltransferase III and VI and α1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.
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  • 3
    ISSN: 1573-6776
    Keywords: fucosyltransferase ; N-acetylglucosaminyltransferase ; P-selectin ; PSGL-1 ; tricistronic vectors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Expression of recombinant glycoproteins carrying the sialyl-LewisX epitope requires host cells equipped with appropriate glycosyltransferases. Chinese hamster ovary cells transfected with tricistronic vectors and encoding the resistance marker gene, neoR, in the third cistron and core 2 N-acetylglucosaminyltransferase or α1,3-fucosyltransferase III or IV in either the first or second cistron, respectively, produced both enzyme activities in a constant ratio. A representative clone was transfected with PSGL-1 (P-selectin glycoprotien ligand 1) and conferred P-selectin-binding activity to PSGL-1.
    Type of Medium: Electronic Resource
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