Efficient preparation of natural and synthetic galactosides with a recombinant beta-1,4-galactosyltransferase-/UDP-4'-gal epimerase fusion protein.
The numerous biological roles of LacNAc-based oligosaccharides have led to an increased demand for these structures for biological studies. In this report, an efficient route for the synthesis of beta-galactosides using a bacterial beta-4-galactosyltransferase/-UDP-4'-gal-epimerase fusion protein is described. The lgtB gene from Neisseria meningitidis and the galE gene from Streptococcus thermophilus were fused and cloned into an expression vector pCW. The fusion protein transfers galactose to a variety of different glucose- and glucosamine-containing acceptors, and utilizes either UDP-galactose or UDP-glucose as donor substrates. A crude lysate from Escherichia coli expressing the fusion protein is demonstrated to be sufficient for the efficient preparation of galactosylated oligosaccharides from inexpensive UDP-glucose in a multigram scale. Lysates containing the fusion protein are also found to be useful in the production of more complex oligosaccharides in coupled reaction mixtures, e.g., in the preparation of sialosides from N-acetylglucosamine. Thus, bacterially expressed fusion protein is well suited for the facile and economic preparation of natural oligosaccharides and synthetic derivatives based on the lactosamine core.
Department of Molecular Biology, The Scripps Research Institute, MEM-L71, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
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Blixt
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Brown
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Schur
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Wakarchuk
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Paulson
D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005697:Galactosides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013291:Streptococcus / Q000201:enzymology; D014534:UDPglucose 4-Epimerase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D005697:Galactosides; D011993:Recombinant Fusion Proteins; D009237:N-Acetyllactosamine Synthase; D014534:UDPglucose 4-Epimerase; C020551:galactose epimerase; D014532:Uridine Diphosphate Glucose