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

  • Liam Doyle
  • Olga G. Ovchinnikova
  • Chris WhitfieldEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Microbial glycobiology Capsular polysaccharide Glycosyltransferase In vitro biochemical reaction Fluorescently-tagged synthetic acceptor 

Notes

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).

References

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Liam Doyle
    • 1
  • Olga G. Ovchinnikova
    • 1
  • Chris Whitfield
    • 1
    Email author
  1. 1.Department of Molecular and Cellular BiologyUniversity of GuelphGuelphCanada

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