Evidence for redox forms of the <i>Aequorea</i> green fluorescent protein

Inouye, Satoshi; Tsuji, Frederick I.

doi:10.1016/0014-5793(94)00859-0

Cited by 139 publications

(95 citation statements)

References 21 publications

(33 reference statements)

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“…3). When these samples were further exposed to either dithionite or FeSO 4 , the fluorescence disappeared and then returned upon exposure to air as previously described for the wild type protein exposed to reducing and oxidizing environments [31]. CE experiments were therefore undertaken to determine whether GFP undergoes oxidative/ reductive damage elsewhere on the protein following treatment with O 2 •− .…”

Section: Resultsmentioning

confidence: 99%

Quenching of superoxide radicals by green fluorescent protein

Bou-Abdallah

Chasteen

Lesser

2006

Biochimica et Biophysica Acta (BBA) - General Subjects

161

144

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Green fluorescent protein (GFP) is a widely used in vivo molecular marker. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O 2 •− ) and exhibits SOD-like activity by competing with cytochrome c for reaction with O 2 •− . When exposed to high amounts of O 2 •− the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.

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Section: Resultsmentioning

confidence: 99%

Quenching of superoxide radicals by green fluorescent protein

Bou-Abdallah

Chasteen

Lesser

2006

Biochimica et Biophysica Acta (BBA) - General Subjects

161

144

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“…Although the mechanism is still unclear, GFP must be modified post-translationally to acquire fluorescent activity. Inouye and Tsuji [22] have reported that the fluorescent intensity of intact dequorea GFP expressed in E. coli cells increases in harvested cells stored overnight at room temperature. Lim et al [23] have also reported the thermosensitive formation of fluorescent GFP in Saccharomyces cerevisiae.…”

Section: Construction and Expression Of Pa-gfpmentioning

confidence: 99%

Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting

et al. 1996

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Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coil The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening.

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“…No additive (calcium, a prosthetic group) is necessary for Aequorin to emit light 4,8,9,12,14,15,16 .…”

Section: Introductionmentioning

confidence: 99%

“…Both groups emit a visible green light in the maximum emission peak (505-509 nm) 14,15,17,18,19,20,21 . The composition of GFP, an 11-stranded β-barrel wrapped around an α-helix, is its most distinctive feature.…”

Section: Introductionmentioning

confidence: 99%

Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

Brito

Carvalho

Zonari

et al. 2013

Braz. J. Vet. Res. Anim. Sci.

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Genotype and expression of the enhanced green fluorescent protein in LEW-Tg (EGFP) F455.5/Rrrc rats AbstractThe Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/ Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary. Keywords: Enhanced green fluorescent protein. EGFP. Autofluorescence. Difference in expression. ResumoA proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.Palavras-chave: Proteína verde fluorescente melhorada. (EGFP). Autofluorescência. Diferença de expressão.

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Evidence for redox forms of the Aequorea green fluorescent protein

Cited by 139 publications

References 21 publications

Quenching of superoxide radicals by green fluorescent protein

Quenching of superoxide radicals by green fluorescent protein

Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting

Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

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