Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein

• Published in: The Journal of biological chemistry Vol. 273; no. 14; pp. 8212 - 8216
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 03-04-1998
• Subjects: Aequorea victoria; Fluorescence; Gene Transfer Techniques; Genes, Reporter; Green Fluorescent Proteins; Humans; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Point Mutation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.14.8212

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.