Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein
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 pr...
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| Published in: | The Journal of biological chemistry Vol. 273; no. 14; pp. 8212 - 8216 |
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| Main Authors: | , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
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American Society for Biochemistry and Molecular Biology
03-04-1998
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| 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 âBFPâ 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 37â°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. |
|---|---|
| AbstractList | 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 degree 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. 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 âBFPâ 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 37â°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. 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 degreesC 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. |
| Author | David W. Piston Paul A. Kitts George H. Patterson Parisa Sinai Robert Chervenak Lonnie Lybarger Steven R. Kain Yih-Tai Chen Te-Tuan Yang Gisele Green |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/9525926$$D View this record in MEDLINE/PubMed |
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| Snippet | The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein... |
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| SubjectTerms | Aequorea victoria Fluorescence Gene Transfer Techniques Genes, Reporter Green Fluorescent Proteins Humans Luminescent Proteins - chemistry Luminescent Proteins - genetics Point Mutation |
| Title | Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein |
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