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 twofold) 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 twofold) 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 sixfold increased fluorescence signal.
Highlights
The strength of protein expression can be influenced by many factors including outside factors such as culture conditions, or inside factors as codon usage or transcription/translation system (Ullrich et al, 2015)
The constructs used for transient transfection contained the promoter:green fluorescent protein (GFP) fusion and a normalization cassette consisting of the 2x 35S promoter, the mCherry gene sequence and a 35S terminator (Figure 1)
We selected the 2x 35S promoter because it is widely used in the P. patens community and shows a medium gene expression in P. patens, which makes it a good candidate for comparisons with new promoters
Summary
The strength of protein expression can be influenced by many factors including outside factors such as culture conditions, or inside factors as codon usage or transcription/translation system (Ullrich et al, 2015). Both promoter sequence and coding sequence can be optimized to improve final protein accumulation. The same study included endogenous 5 sequences of the genes α1,3-fucosyltransferase and β1,2-xylosyltransferase (fuc-t, xyl-t) that were further characterized via deletion constructs, with the 5 -fuc-t showing almost double activity as compared to the single CaMV 35S promoter. Other endogenous promoters were used, e.g., different tubulin (Jost et al, 2005) or actin promoters (Weise et al, 2006), all of which showed stronger expression than the CaMV 35S promoter, that was later shown to yield only mediocre expression in P. patens, especially in the dark (Saidi et al, 2009)
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