Creation of Circularly Permutated Yellow Fluorescent Proteins Using Fluorescence Screening and a Tandem Fusion Template |
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Authors: | Jason Jui-Hsuan Chiang Isaac Li Kevin Truong |
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Institution: | (1) Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, M5S 3G9 Toronto, Ont., Canada;(2) Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, M5S 3G4 Toronto, Ont., Canada |
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Abstract: | By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence
resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative
fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach
to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid
because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP
variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong
similarity to YFP and therefore can be used in replacement.
Revisions requested 27 October 2005; Revisions received 23 December 2005 |
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Keywords: | circular permutation fluorescent protein protein engineering |
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