CASTDan:Abstract17
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.
