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

Type de document :

Article dans une revue scientifique: Article original

DOI :

10.3390/ijms19020371

URL permanente :

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

Titre :

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

Auteur(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

Titre de la revue :

International Journal of Molecular Sciences

Nom court de la revue :

IJMS

Numéro :

Pagination :

371

Date de publication :

2018-01-26

ISSN :

1661-6596

Discipline(s) HAL :

Sciences du Vivant [q-bio]

Résumé en anglais : [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 ...
Lire la suite >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.Lire moins >

Langue :

Anglais

Audience :

Internationale

Vulgarisation :

Non

Établissement(s) :

CNRS
ENSCL
Université de Lille

Collections :

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

Date de dépôt :

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

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Ce document est diffusé sous licence Attribution 3.0 United States

Numéro de version	Lien	Date de modification
2	20.500.12210/57774*	2023-01-04T09:49:20Z
1	20.500.12210/57774.1	2021-11-16T08:23:28Z

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