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  • Cloning of the Lipooligosaccharide a-2,3-Sialytransferase from the Bacterial Pathogens Neisseria Meningitidis and Neisseria Goorrhoeae

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    Author(s)
    Gilbert, M
    Watson, DC
    Cunningham, AM
    Jennings, MP
    Young, NM
    Wakarchuk, WW
    Griffith University Author(s)
    Jennings, Michael P.
    Year published
    1996
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    Abstract
    The genes encoding the α-2,3-sialyltransferases involved in lipooligosaccharide biosynthesis from Neisseria meningitidis and Neisseria gonorrhoeae have been cloned and expressed in Escherichia coli. A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransferase acceptor and capillary electrophoresis was used to screen a genomic library of N. meningitidis MC58 L3 in a “divide and conquer” strategy. The gene, denoted lst, was found on a 2.0-kilobase fragment of DNA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding lst genes from N. meningitidis ...
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    The genes encoding the α-2,3-sialyltransferases involved in lipooligosaccharide biosynthesis from Neisseria meningitidis and Neisseria gonorrhoeae have been cloned and expressed in Escherichia coli. A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransferase acceptor and capillary electrophoresis was used to screen a genomic library of N. meningitidis MC58 L3 in a “divide and conquer” strategy. The gene, denoted lst, was found on a 2.0-kilobase fragment of DNA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding lst genes from N. meningitidis 406Y L3, N. meningitidis M982B L7, and N. gonorrhoeae F62. Functional sialyltransferase was produced from the genes derived from both L3 N. meningitidis strains and the N. gonorrhoeae F62. However, the N. meningitidis M982B L7 gene contained a frameshift mutation that renders it inactive. The expression of the lst gene was easily detected using the enzyme assay, and the protein expression could be detected when an immunodetection tag was added to the COOH-terminal end of the protein. Using the synthetic acceptor N-acetyllactosamine-aminophenyl-(6-(5-(fluorescein-carboxamido)-hexanoic acid amide), the α-2,3 specificity of the enzyme was confirmed by NMR examination of the reaction product. The enzyme could also use synthetic acceptors with lactose or galactose as the saccharide portion. This study is the first example of the cloning, expression, and examination of α-2,3-sialyltransferase activity from a bacterial source.
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    Journal Title
    Journal of Biological Chemistry
    Volume
    271
    Issue
    45
    Publisher URI
    http://www.jbc.org/content/271/45/28271.short
    Copyright Statement
    This research was originally published in Journal of Biological Chemistry (JBC). Gilbert, et. al., Cloning of the Lipooligosaccharide a-2,3-Sialytransferase from the Bacterial Pathogens Neisseria Meningitidis and Neisseria Goorrhoeae, Journal of Biological Chemistry (JBC), 271, 28271-28276., 1996. Copyright the American Society for Biochemistry and Molecular Biology. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive version.
    Subject
    Chemical sciences
    Biological sciences
    Biomedical and clinical sciences
    Publication URI
    http://hdl.handle.net/10072/120906
    Collection
    • Journal articles

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