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Fluorogen-activating single-chain antibodies for imaging cell surface proteins
Christopher Szent-Gyorgyi1, Brigitte F Schmidt, Brigitte A Schmidt
1Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. css@andrew.cmu.edu
Nature Biotechnology
|December 25, 2007
Summary
Researchers developed novel protein reporters called fluorogen activating proteins (FAPs) that generate bright fluorescence from non-fluorescent molecules. This breakthrough enhances live-cell imaging by enabling thousands-fold fluorescence increases with high affinity binding.
Area of Science:
- Biotechnology
- Molecular Biology
- Cellular Imaging
Background:
- Genetically encoded fluorescent proteins revolutionized live-cell imaging.
- Existing methods rely on fluorescent proteins or peptide tags binding exogenous fluorophores.
Purpose of the Study:
- To develop novel protein reporters that generate fluorescence from non-fluorescent molecules (fluorogens).
- To create a new class of probes for enhanced live-cell imaging.
Main Methods:
- Screening a library of human single-chain antibodies (scFvs) to isolate eight unique fluorogen activating proteins (FAPs).
- Utilizing thiazole orange and malachite green derivatives as fluorogens.
- Displaying FAPs on yeast and mammalian cell surfaces.
Main Results:
- FAPs bind fluorogens with nanomolar affinity, achieving thousands-fold increases in fluorescence.
- Achieved brightness levels comparable to traditional fluorescent proteins.
- Demonstrated spectral variation by combining different FAPs and fluorogens.
- Enabled visualization within mammalian cells using membrane-permeant or impermeant fluorogens.
Conclusions:
- The FAP technique offers a powerful new tool for live-cell imaging.
- This method is extensible to various nonfluorescent dyes, expanding its applicability.
- FAPs provide a versatile platform for generating bright, tunable fluorescence signals.
