Fluorescence intensity- and lifetime-based glucose sensing using an engineered high-Kd mutant of glucose/galactose-binding protein.
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.
Khan
Faaizah
F
Diabetes Research Group, King's College London School of Medicine, Guy's Hospital, London SE1 1UL, UK.
Saxl
Tania E
TE
Pickup
John C
JC
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D019943:Amino Acid Substitution; D015374:Biosensing Techniques; D001786:Blood Glucose / Q000032:analysis*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003920:Diabetes Mellitus / Q000175:diagnosis; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D001786:Blood Glucose; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D020033:Protein Isoforms; C023550:galactose-binding protein; D015081:2-Naphthylamine