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ID 57798
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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 Kakenhi 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 Kakenhi publons
Abstract
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.
Published Date
2019-3-18
Publication Title
Scientific Reports
Volume
volume9
Publisher
Nature Publishing Group
Start Page
4722
ISSN
2045-2322
Content Type
Journal Article
language
英語
OAI-PMH Set
岡山大学
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publisher
PubMed ID
DOI
Web of Science KeyUT
Related Url
isVersionOf https://doi.org/10.1038/s41598-019-41309-8
License
https://creativecommons.org/licenses/by/4.0/
Funder Name
Japan Society for the Promotion of Science
助成番号
15H05370
17H03644
25891017