Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells = 효모의 당 대사를 감지하는 고감도 FRET

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Title
Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells = 효모의 당 대사를 감지하는 고감도 FRET
Author(s)
Jae Seok Ha; Jae Jun Song; Y M Lee; Su Jin Kim; Jung Hoon Sohn; C S Shin; Seung Goo Lee
Bibliographic Citation
Applied and Environmental Microbiology, vol. 73, no. 22, pp. 7408-7414
Publication Year
2007
Abstract
A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker1-maltose-binding protein-linker2-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).
ISSN
0099-2240
Publisher
Amer Soc Microb
DOI
http://dx.doi.org/10.1128/AEM.01080-07
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > 1. Journal Articles
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