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Int. J. Mol. Sci. 2018, 19(2), 371; doi:10.3390/ijms19020371

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

1
Department of Bioengineering, Graduate School of Science and Engineering, Yamagata University, 992-8510 Yonezawa, Japan
2
Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, KU Leuven, Celestijnenlaan 200g Box 2403, 3001 Leuven, Belgium
3
Laboratoire de Spectrochimie Infrarouge et Raman, Université de Lille, CNRS, UMR 8516, LASIR, F59 000 Lille, France
4
Department of Bio-System Engineering, Graduate School of Science and Engineering, Yamagata University, 992-8510 Yonezawa, Japan
*
Author to whom correspondence should be addressed.
Received: 20 December 2017 / Revised: 15 January 2018 / Accepted: 24 January 2018 / Published: 26 January 2018
(This article belongs to the Special Issue Fluorescent Proteins)
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Abstract

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. View Full-Text
Keywords: fluorescent protein; expression cloning; reading frame; frameshift fluorescent protein; expression cloning; reading frame; frameshift
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Horiuchi, Y.; Laskaratou, D.; Sliwa, M.; Ruckebusch, C.; Hatori, K.; Mizuno, H.; Hotta, J.-I. Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense. Int. J. Mol. Sci. 2018, 19, 371.

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