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High-throughput screening methodology for the directed evolution of glycosyltransferases

Academic Article
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Overview

authors

  • Aharoni, A.
  • Thieme, K.
  • Chiu, C. P. C.
  • Buchini, S.
  • Lairson, Luke
  • Chen, H. M.
  • Strynadka, N. C. J.
  • Wakarchuk, W. W.
  • Withers, S. G.

publication date

  • 2006

journal

  • Nature Methods  Journal

abstract

  • Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of new oligosaccharides holds great potential for the development of the field of glycobiology. However, engineering of GTs by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of a new fluorescence-based high-throughput screening (HTS) methodology for the directed evolution of sialyltransferases (STs). Using this methodology, we detected the formation of sialosides in intact Escherichia coli cells by selectively trapping the fluorescently labeled transfer products in the cell and analyzing and sorting the resulting cell population using a fluorescence-activated cell sorter (FACS). We screened a library of >10(6) ST mutants using this methodology and found a variant with up to 400-fold higher catalytic efficiency for transfer to a variety of fluorescently labeled acceptor sugars, including a thiosugar, yielding a metabolically stable product.

subject areas

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Biological Assay
  • Biotechnology
  • Enzyme Activation
  • Escherichia coli
  • Evolution, Molecular
  • Flow Cytometry
  • Glycosyltransferases
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Array Analysis
  • Sequence Analysis, Protein
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Identity

International Standard Serial Number (ISSN)

  • 1548-7091

Digital Object Identifier (DOI)

  • 10.1038/nmeth899

PubMed ID

  • 16862135
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Additional Document Info

start page

  • 609

end page

  • 614

volume

  • 3

issue

  • 8

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