Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein*
- Te-Tuan Yang‡,
- Parisa Sinai‡,
- Gisele Green‡,
- Paul A. Kitts‡,
- Yih-Tai Chen§,
- Lonnie Lybarger¶,
- Robert Chervenak¶,
- George H. Patterson‖,
- David W. Piston‖ and
- Steven R. Kain‡**
- From the ‡Cell Biology Group, Clontech Laboratories, Inc., Palo Alto, California 94303, the §Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, the ¶Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, Louisiana 71130, and the ‖Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232
Abstract
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described “BFP” reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 °C and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵** To whom correspondence should be addressed: Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303-4230. Tel.: 650-424-8222, ext. 1469; Fax: 650-354-0776; E-mail: srkain{at}clontech.com.
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↵1 The abbreviations used are: GFP, green fluorescent protein; BFP, blue fluorescent protein; wtGFP, wild-type green fluorescent protein; EBFP, blue emission variant of GFP; EGFP, red-shifted emission variant of GFP; QY, quantum yield.
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- Received August 29, 1997.
- Revision received January 14, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
