Hello everyone! I'm currently trying to increase the expression of my fusion protein in a C. elegans animal model. After reading papers, I found two possible methods to try: (1) codon-optimization and (2) intron-mediated enhancement of gene expression.
Regarding intron-mediated enhancement, a paper (see link below) suggested that inserting introns in between the sequence of EGFP increased its expression by ~80%. However, their experiment only studied the expression of fluorescent proteins and they did not fuse any other proteins downstream of EGFP.
https://www.nature.com/articles/s41598-019-45517-0#Sec11
I am wondering whether putting EGFP (with introns) at the N-terminal of my fusion protein would also increase the expression of all other proteins downstream of it, or will I need to experiment on intron insertion throughout the fusion protein too?
TIA!
Sure, this is possible in this experimantal setting. However, it should be experimentally evaluated (e.g. via knockdown and corresponding rescue experiments).
In theory, it would. The cell wouldn't necessarily have a way to "know" that your fusion partners are supposed to be separate. If you design the construct well, it can appear to the cell just like any old multi-domain protein.
In practice, cell biology isn't like computer programming. Every construct you plug in can have completely unanticipated effects. The best strategy is designing multiple constructs, as different from each other as your research goals allow, and testing them all out.
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