Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense
AbstractExpression 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
Scifeed alert for new publicationsNever miss any articles matching your research from any publisher
- Get alerts for new papers matching your research
- Find out the new papers from selected authors
- Updated daily for 49'000+ journals and 6000+ publishers
- Define your Scifeed now
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.
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. International Journal of Molecular Sciences. 2018; 19(2):371.Chicago/Turabian Style
Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-ichi. 2018. "Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense." Int. J. Mol. Sci. 19, no. 2: 371.
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.