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ID 57798
フルテキストURL
著者
Nakatani, Takahiro Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
Yasui, Norihisa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University ORCID Kaken ID publons
Tamura, Issei Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
Yamashita, Atsuko Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kaken ID publons
抄録
Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the epsilon-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems.
発行日
2019-3-18
出版物タイトル
Scientific Reports
9巻
出版者
Nature Publishing Group
開始ページ
4722
ISSN
2045-2322
資料タイプ
学術雑誌論文
言語
English
OAI-PMH Set
岡山大学
論文のバージョン
publisher
PubMed ID
DOI
Web of Science KeyUT
関連URL
isVersionOf https://doi.org/10.1038/s41598-019-41309-8
ライセンス
https://creativecommons.org/licenses/by/4.0/
助成機関名
日本学術振興会
助成番号
15H05370
17H03644
25891017