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  Combination of the endogenous lhcsr1 promoter and codon usage optimization boosts protein expression in the Moss Physcomitrella patens

Hiss, M., Schneider, L., Grosche, C., Barth, M. A., Neu, C., Symeonidi, A., et al. (2017). Combination of the endogenous lhcsr1 promoter and codon usage optimization boosts protein expression in the Moss Physcomitrella patens. Frontiers in Plant Science, 8: 1842. doi:10.3389/fpls.2017.01842.

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 Creators:
Hiss, Manuel, Author
Schneider, Lucas, Author
Grosche, Christopher, Author
Barth, Melanie A., Author
Neu, Christina, Author
Symeonidi, Aikaterini, Author
Ullrich, Kristian K.1, Author           
Perroud, Pierre-François, Author
Schallenberg-Rüdinger, Mareike, Author
Rensing, Stefan A., Author
Affiliations:
1Department Evolutionary Genetics, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_1445635              

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Free keywords: Physcomitrella patens; codon usage; chlorophyll a/b binding protein; promoter; codon bias; green fluorescent protein (GFP); lhcsr1; fluorescence normalization
 Abstract: The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g. selectable markers, fluorescent proteins or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein LHCSR1 drives expression of GFP in protoplasts significantly (more than 2-fold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than 2-fold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed 6-fold increases fluorescence signal.

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Language(s): eng - English
 Dates: 2017-08-192017-10-192017-10-312017
 Publication Status: Issued
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 Identifiers: DOI: 10.3389/fpls.2017.01842
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Funding organization : German Federal Ministry of Education and Research (Freiburg Initiative for Systems Biology, 0313921 to SR) (0313921)

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Title: Frontiers in Plant Science
  Abbreviation : Front. Plant Sci.
Source Genre: Journal
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Publ. Info: Lausanne : Frontiers Media
Pages: 11 Volume / Issue: 8 Sequence Number: 1842 Start / End Page: - Identifier: ISSN: 1664-462X
CoNE: https://pure.mpg.de/cone/journals/resource/1664462X