A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution

Nat Protoc. 2008;3(3):357-62. doi: 10.1038/nprot.2007.538.

Abstract

This protocol details the application of a high-throughput fluorescence-based screen, in conjunction with error-prone PCR/saturation mutagenesis, for altering the proficiency and/or promiscuity of a secondary metabolite glycosyltransferase (GT) via directed evolution. Given the structural and mechanistic similarities among secondary metabolite-associated GTs, this approach may provide a template for engineering other members of the GT-B superfamily.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / genetics
  • Biomedical Engineering / methods*
  • Directed Molecular Evolution / methods*
  • Escherichia coli / genetics
  • Fluorescence
  • Gene Library
  • Glucosyltransferases / genetics
  • Glycosylation
  • Glycosyltransferases / analysis*
  • Glycosyltransferases / metabolism
  • Mutagenesis / genetics
  • Mutation / genetics
  • Streptomyces antibioticus / enzymology

Substances

  • Bacterial Proteins
  • Glycosyltransferases
  • Glucosyltransferases
  • OleD protein, Streptomyces antibioticus