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Bacterial Polysaccharides pp 151–159Cite as

Utilization of Fluorescently Tagged Synthetic Acceptor Molecules for In Vitro Characterization of a Dual-Domain Glycosyltransferase Enzyme, KpsC, from Escherichia coli

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1954))

Abstract

The incorporation of fluorescent tags into synthetic acceptor molecules for in vitro biochemical assays allows quick and easy detection of enzyme activity. Reaction products can be separated via thin-layer chromatography and visualized under UV light for rapid detection of reaction progress. Subsequent structural analysis of these reaction products through the use of NMR spectroscopy and mass spectrometry allows for complete functional characterization of enzyme activity. Here we describe an application of this technique which was previously used to functionally characterize a dual-domain glycosyltransferase enzyme, KpsC, involved in capsular polysaccharide biosynthesis in Escherichia coli.

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References

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Acknowledgments

We thank Bo-Shun Huang, Matthew S. Kimber, and Todd L. Lowary for their contributions to this project originally published as reference 6. This work was supported by operating funding from the Canadian Institutes of Health Research, the National Science and Engineering Research Council of Canada, and the Canadian Glycomics Network (GlycoNet, National Centres of Excellence Program).

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

    Liam Doyle, Olga G. Ovchinnikova & Chris Whitfield

Authors
  1. Liam Doyle
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  2. Olga G. Ovchinnikova
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  3. Chris Whitfield
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Corresponding author

Correspondence to Chris Whitfield .

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Editors and Affiliations

  1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Inka Brockhausen

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Doyle, L., Ovchinnikova, O.G., Whitfield, C. (2019). Utilization of Fluorescently Tagged Synthetic Acceptor Molecules for In Vitro Characterization of a Dual-Domain Glycosyltransferase Enzyme, KpsC, from Escherichia coli. In: Brockhausen, I. (eds) Bacterial Polysaccharides. Methods in Molecular Biology, vol 1954. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9154-9_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9154-9_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9153-2

  • Online ISBN: 978-1-4939-9154-9

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