title: Dual-color-emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy.
abstract: We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long-pass emission filter with single-wavelength excitation, which is technically easier to use than dual-emission fluorescent proteins that require dual-wavelength excitation.
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Kuboyama 2-3, Hachioji, Tokyo 192-8512, Japan. katsunori_ogo@ot.olympus.co.jp
authors.forename: Katsunori
authors.initials: K
authors.lastname: Ogoh
authors.forename: Takashi
authors.initials: T
authors.lastname: Kinebuchi
authors.forename: Mariko
authors.initials: M
authors.lastname: Murai
authors.forename: Takeo
authors.initials: T
authors.lastname: Takahashi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
authors.forename: Hirobumi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000737:chemistry*; D002460:Cell Line; D003116:Color; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data
chemical_list: D007202:Indicators and Reagents; D049452:Green Fluorescent Proteins
keywords: GFP, dual-color emission; fluorescence microscopy; pH indicator; sea cactus