Engineering a Reagentless Glucose Sensor from the Periplasmic Binding Protein of E. coli

Archana Sriram



Glucose sensing remains a very active research effort in the field of diagnostics, as well as bioprocess monitoring. Diabetes is a chronic disease characterized by the body's inability to produce or utilize insulin, thereby leading to uncontrolled glucose levels in blood and tissues. This disease affects 6.2% of the total population and 20.1% of those aged 65 years or older in the United States. Thus, the cost for the control of this disease and the prevention of its complications can be as high as $132 billion dollars annually. Diabetes results in long-term health consequences, including cardiovascular disease and blindness. Glucose levels in blood and plasma are used as a clinical indicator of diabetes. For the above reasons, there is continued interest in new and improved methods of glucose monitoring for clinical purposes.

Coming to the bioprocessing aspect, glucose is the major carbon and energy source in cellular metabolism. The lack of glucose in the medium will severely limit cell growth and product yield in industrial bioprocess applications. However, excessive glucose can also be detrimental, leading to lactate formation via the glycolytic pathway leading to lactate formation, which will in turn inhibit cellular metabolism. Therefore, glucose monitoring and control is important for healthy growth of cells and maximum product formation in bioprocess.


Our research mainly focuses on developing a novel set of proteins for sensing applications. The key element in designing these biosensors are the soluble proteins found in the periplasmic space of the gram-negative bacteria such as Escherichia coli. The main focus is on glucose/galactose binding protein (GBP) that primarily recognizes glucose. Three mutants of this protein E149C, L255C, E149C/L255C have been generated, where cysteine residues have been introduced in to the domain of the protein. The cysteine residues are then labeled using fluorescent probes so that the mutated protein can be used for glucose quantification. Additionally, immobilization of these proteins on chemically modified glass surfaces can be used to sense presence of glucose in biological samples. On immobilization, the ligand binding process is reversible and hence these mutated proteins can be used repeatedly.