CASTDan:Abstract17

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Kostov, Y.,Tolosa L.,Ge X., Lakowiczb J.R., Rao G (2003) Dual-Emitting Biosensors for Glucose and Glutamine from Genetically Engineered E. coli Binding Proteins. Genetically Engineered and Optical Probes for Biomedical Application. Vol. 4967

Abstract:


Glucose is the major source of carbon, and glutamine is the major source of nitrogen in cell culture media. Thus, glucose and glutamine monitoring are important in maintaining optimal conditions in industrial bioprocesses. Here we report reagentless glucose and glutamine sensors using the E. coli glucose binding protein (GBP) and the glutamine binding protein (GlnBP). Both of these proteins are derived from the permease system of the gram-negative bacteria. The Q26C variant of GBP was labeled at the 26-position with anilino-naphthalene sulfonate (ANS), while the S179C variant of GlnBP was labeled at the 179-position with acrylodan. The ANS and acrylodan emissions are quenched in the presence of glucose and glutamine, respectively. The acrylodan-labeled GlnBP was labeled at the N-terminal with ruthenium bis-(2,2’-bipyridyl)-1,10-phenanthroline-9-isothiocyanate. The ruthenium acts as a non-responsive longlived reference. The apparent binding constant, Kd’, of 8.0 μM glucose was obtained from the decrease in intensity of ANS in GBP. The reliability of the method in monitoring glucose during yeast fermentation was determined by comparison with the YSI Biochemistry Analyzer. The apparent binding constant, Kd’, of 0.72 μM glutamine was calculated from the ratio of emission intensities of acrylodan and ruthenium (I515/I610) in GlnBP. The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of + 0.02 μM. The conversion of the GBP into a similar ratiometric sensor was described.


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