We discovered that some isolated eel skeletal muscle cells exhibited green fluorescence under a fluorescence stereomicroscope, and we successfully isolated a novel fluorescent protein from the eel muscle homogenate. The protein was a monomer with a molecular mass of 16.5–17 kDa and showed minor and major peaks at 280 and 493 nm, respectively, in the absorption spectrum. The molar extinction coefficient at 493 nm was 41,300 M−1 cm−1 and A280/A493 was 0.083. Excitation and emission spectra of the protein showed maxima at 493 and 527 nm, respectively. Heat treatment at 95°C for 10 min or 5% trichloroacetic acid treatment of the protein caused aggregation of the protein but did not release any fluorescent components such as FAD into the supernatant after centrifugation. Fluorescence of the protein remained after native PAGE, but not after SDS-PAGE. These results indicate that the purified fluorescent protein is not a flavoprotein, and that its fluorescent chromophore is a covalently bound one, such as green fluorescent protein (GFP) from jellyfish Aequoria victoria, but that its fluorescence requires its native conformation within the protein. Based on these results, we can conclude that the fluorescent protein obtained from eel skeletal muscle is a novel GFP-like protein.
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We thank Dr. Kobayashi, Takara Bio Inc., for helpful suggestions. This work was supported in part by a Grant-in-Aid for Basic Research B (10460095) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and was also supported by Takara Bio Inc. We also thank Messrs Adachi and Honda for technical support.
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Hayashi, S., Toda, Y. A novel fluorescent protein purified from eel muscle. Fish Sci 75, 1461–1469 (2009). https://doi.org/10.1007/s12562-009-0176-z
- Fluorescent protein
- GFP-like protein
- Isolated muscle cells
- Skeletal muscle