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Proceedings Paper

Dual-emitting biosensors for glucose and glutamine from genertically engineered E. coli binding proteins
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Paper 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 long-lived 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.

Paper Details

Date Published: 12 September 2003
PDF: 7 pages
Proc. SPIE 4967, Genetically Engineered and Optical Probes for Biomedical Applications, (12 September 2003); doi: 10.1117/12.485718
Show Author Affiliations
Leah Tolosa, Univ. of Maryland, Baltimore County (United States)
Xudong Ge, Univ. of Maryland, Baltimore County (United States)
Yordan Kostov, Univ. of Maryland, Baltimore County (United States)
Joseph R. Lakowicz, Ctr. for Fluorescence Spectroscopy (United States)
University of Maryland School of Medicine (United States)
Govind Rao, Univ. of Maryland, Baltimore County (United States)


Published in SPIE Proceedings Vol. 4967:
Genetically Engineered and Optical Probes for Biomedical Applications
Darryl J. Bornhop; Alexander P. Savitsky; Ramesh Raghavachari; Samuel I. Achilefu, Editor(s)

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