Genetic Engineering of an Allosterically Based Glucose Indicator Protein for Continuous Glucose Monitoring by Fluorescence Resonance Energy Transfer

• Published in: Analytical chemistry (Washington) 2003-07, Vol.75 (14), p.3451-3459
• Main Authors: Ye, Kaiming, Schultz, Jerome S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biosensing Techniques; Chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - isolation & purification; Exact sciences and technology; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; General, instrumentation; Genetic Engineering; glucose; Glucose - analysis; glucose indicator protein; Indicators and Reagents; Molecular Sequence Data; Protein Binding; Protein Conformation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Spectrometric and optical methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac034022q

Real-time monitoring of blood glucose could vastly reduce a number of the long-term complications associated with diabetes. In this article, we present a novel approach that relies on a glucose-binding protein engineered such that a 20% reduction in fluorescence due to the fluorescence resonance energy transfer occurs as a result of glucose binding. This change in fluorescence provides a signal for the optical detection of glucose. The novel glucose indicator protein (GIP) was created by fusing two fluorescent reporter proteins (green fluorescent proteins) to each end of an Escherichia coli glucose-binding protein in such a manner that the spatial separation between the fluorescent moieties changes when glucose binds, thus generating a distinct optical signal that can be used for glucose detection. By placing the GIP within a dialysis hollow fiber sensor, a microsensor has been developed for continuous monitoring of glucose. The sensor had a response time to sudden glucose changes within 100 s and was reversible. The sensor was shown to have an optional range on the order of 10 μM of glucose.