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OwnerRisto Renkonen, Pirkko Mattila +8inventors
InventorsRisto Renkonen, Pirkko Mattila, Laura Hirvas, Solveing Hortling, Tuula Kallioinen, Sirkka-Liisa Kauranen, Nina Jarvinen, Minna Maki, Jaana Niittymaki, Jarkko Rabina
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Use of recombinant enzymes for the preparation of GDP-L-fucose and fucosylated glycans is disclosed. GDP-L-fucose functions as a fucose donor in the biosynthetic route leading to the fucosylated glycans, which have therapeutic utility. A process for preparing GDP-L-fucose and fucosylated glycans, and means useful in the process are provided. Said means include enzymes, chimeric enzymes, DNA sequences, genes, vectors and host cells. An assay for the determination of GDP-fucose and fucosyltransferase, and a test kit therefore are also provided.
A process for preparing GDP-L-fucose from GDP-D-mannose, wherein the GDP-L-fucose is prepared from inherent GDP-D-mannose by cultivating yeast or mold cells, which have been transformed with a DNA sequence coding for GDP-mannose-4,6-dehydratase (GMD) and a DNA sequence coding for GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GFS) to functionally express said enzymes, and by recovering the GDP-L-fucose formed from the GDP-D-mannose inherent in said cells.
A chimeric enzyme which is a chimeric molecule of GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GFS).
A vector comprising a DNA sequence encoding a chimeric molecule of GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GFS).
A process for preparing GDP-L-fucose, wherein said compound is prepared from GDP-D-mannose through the de novo pathway using at least one of the recombinant enzymes selected from the group consisting of GDP-mannose-4,6-dehydratase (GMD) of Helicobacter pylori and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GFS) of Helicobacter pylori.
An isolated DNA sequence encoding Helicobacter pylori GDP-mannose-4,6-dehydratase (GMD).
An isolated DNA sequence encoding Helicobacter pylori GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GFS).
A process for preparing GDP fucose, wherein said compound is prepared from L-fucose through the salvage pathway using at least one of the recombinant enzymes selected from the group consisting of fuco-1-kinase (FK) and GDP-fucose-pyrophosphorylase (PP).
An isolated DNA sequence encoding murine or human fuco kinase (FK).
An isolated DNA sequence encoding rat or murine GDP fucose pyrophosphorylase (PP).
An isolated DNA sequence encoding Helicobacter felis or rat α-1,3-fu-cosyltransferase.
An assay for the determination of GDP Fucose or fucosyltransferase said assay comprising employing biotinylated carbohydratepolyacrylamide conjugates, streptavidin and time resolved fluorometric detection.
Dependencies are inferred from the claim text.