A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling.
We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.
Karasawa
Satoshi
S
Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN 2-1 Hirosawa, Wako-City, Saitama 351-0198, Japan.
Araki
Toshio
T
Yamamoto-Hino
Miki
M
Miyawaki
Atsushi
A
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins