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Fluorescence intensity- and lifetime-based glucose sensing using an engineered high-Kd mutant of glucose/galactose-binding protein.

Abstract
We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213R/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on the addition of glucose, with a binding constant (K(d)) of 11 mM, an operating range of approximately 1-100 mM, and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that the GBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sensor for in vivo glucose monitoring in diabetes.
AuthorsFaaizah Khan, Tania E Saxl, John C Pickup
JournalAnalytical biochemistry (Anal Biochem) Vol. 399 Issue 1 Pg. 39-43 (Apr 01 2010) ISSN: 1096-0309 [Electronic] United States
PMID19961827 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright 2009 Elsevier Inc. All rights reserved.
Chemical References
  • 6-bromoacetyl-2-dimethylaminonaphthalene
  • Blood Glucose
  • Calcium-Binding Proteins
  • Fluorescent Dyes
  • Monosaccharide Transport Proteins
  • Periplasmic Binding Proteins
  • Protein Isoforms
  • galactose-binding protein
  • 2-Naphthylamine
Topics
  • 2-Naphthylamine (analogs & derivatives, chemistry)
  • Amino Acid Substitution
  • Biosensing Techniques
  • Blood Glucose (analysis)
  • Calcium-Binding Proteins (genetics, metabolism)
  • Diabetes Mellitus (diagnosis)
  • Enzyme Assays (methods)
  • Fluorescent Dyes (chemistry)
  • Humans
  • Monosaccharide Transport Proteins (genetics, metabolism)
  • Mutagenesis, Site-Directed
  • Periplasmic Binding Proteins (genetics, metabolism)
  • Protein Isoforms (genetics, metabolism)

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