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, Steven R. Kain

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118 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)8212-8216
Number of pages5
JournalJournal of Biological Chemistry
Volume273
Issue number14
DOIs
StatePublished - Apr 3 1998
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Yang, T. T., Sinai, P., Green, G., Kitts, P. A., Chen, Y. T., Lybarger, L., Chervenak, R., Patterson, G. H., Piston, D. W., & Kain, S. R. (1998). Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. Journal of Biological Chemistry, 273(14), 8212-8216. https://doi.org/10.1074/jbc.273.14.8212