論文 - 詳細
| RRC ID | 50106 |
|---|---|
| 著者 | Shimozono S, Hosoi H, Mizuno H, Fukano T, Tahara T, Miyawaki A. |
| タイトル | Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer. |
| ジャーナル | Biochemistry |
| Abstract |
Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging. |
| 巻・号 | 45(20) |
| ページ | 6267-71 |
| 公開日 | 2006-5-23 |
| DOI | 10.1021/bi060093i |
| PMID | 16700538 |
| MeSH | Anisotropy Bacterial Proteins / chemistry* Bacterial Proteins / metabolism Fluorescence Resonance Energy Transfer / methods* Green Fluorescent Proteins / chemistry* Green Fluorescent Proteins / metabolism HeLa Cells Humans Kinetics Lasers Luminescent Proteins / chemistry* Luminescent Proteins / metabolism Peptide Hydrolases / metabolism Peptide Hydrolases / pharmacology Photobleaching Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Transfection |
| IF | 2.865 |
| 引用数 | 42 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 2 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 遺伝子材料 | Cy11.5/pRSETB (RDB15706). |