Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-ichi

Document type :

Article dans une revue scientifique: Article original

DOI :

10.3390/ijms19020371

Permalink :

http://hdl.handle.net/20.500.12210/57774

Title :

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

Author(s) :

Horiuchi, Yuki [Auteur]
Yamagata University
Laskaratou, Danai [Auteur]
Catholic University of Leuven = Katholieke Universiteit Leuven [KU Leuven]
Sliwa, Michel [Auteur]

Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement (LASIRE) - UMR 8516
Ruckebusch, Cyril [Auteur]

Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement (LASIRE) - UMR 8516
Hatori, Kuniyuki [Auteur]
Yamagata University
Mizuno, Hideaki [Auteur]
Catholic University of Leuven = Katholieke Universiteit Leuven [KU Leuven]
Hotta, Jun-ichi [Auteur]
Yamagata University

Journal title :

International Journal of Molecular Sciences

Abbreviated title :

IJMS

Volume number :

Pages :

371

Publication date :

2018-01-26

ISSN :

1661-6596

HAL domain(s) :

Sciences du Vivant [q-bio]

English abstract : [en]

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally ...
Show more >Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.Show less >

Language :

Anglais

Audience :

Internationale

Popular science :

Non

Administrative institution(s) :

CNRS
ENSCL
Université de Lille

Collections :

Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement (LASIRE) - UMR 8516

Submission date :

2021-11-16T08:23:28Z
2023-01-04T09:58:06Z

Files

ijms-19-00371.pdf
Version éditeur
Open access
Access the document

Except where otherwise noted, this item's license is described as Attribution 3.0 United States

Version	Link	Modification date
2	20.500.12210/57774*	2023-01-04T09:49:20Z
1	20.500.12210/57774.1	2021-11-16T08:23:28Z

Frame-Insensitive Expression Cloning of ...

BibTeX

CSV

Excel

RIS

Files

Version history

Frame-Insensitive Expression Cloning of ... BibTeX CSV Excel RIS

Files

Version history

Frame-Insensitive Expression Cloning of ...

BibTeX

CSV

Excel

RIS