I am planning to express a recombinant protein in Ecoli and want it secreted extracellular (into the media). I have been reading that Gaussia's sec signal would work in prokaryotes as well. I wonder if the secretion signal is cleaved or not.
Also, do we know if it follows sec or tat pathway?
Any help would be appreciated.
Thank you
My educated guess would be: use a prokaryotic signal peptide and create a corresponding cleavage site (otherwise I do not expect cleavage in E. coli).
Whether it follows the Sec pathway depends on your protein (is it to be expected to fold, does it require a special chaperone and so on).
According to underneath review it has been demonstrates that prokaryotic signal peptides don’t render efficient protein translocation/secretion in mammalian cells. It seems logic to expect similar problems the other way around. Because of this I suggest the above described approach.
Stern, B., Olsen, L. C., Tröße, C., Ravneberg, H., & Pryme, I. F. (2007). Improving mammalian cell factories: The selection of signal peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells. Trends Cell Mol Biol, 2, 1-17.
I agree with Rob Keller's suggestion. While the Gaussia signal sequence might work in E. coli, it likely will not be as efficient as an E.coli optimized signal. Why not use an expression vector specifically designed for secretion to the periplasm, there are many of those around. It would be using the universal Sec system if you are utilizing an N-terminal cleavable signal. .
The only reason, I was going with Gaussia signal is because I want the protein to be secreted outside into extracellular region (into media). Most of the signal peptides secrete the protein only into periplasm. Please correct me if I am wrong.@ Rob Keller Michael J. Benedik
I prefer not to use additives to release the proteins from periplasm
Venkata - unfortunately you are not correct. The signal sequence only engages the machinery for secretion across the first membrane. In eukaryotic cells and in Gram positive bacteria, where there is only a single membrane enclosing the cytoplasm, this results in extracellular secretion. But gram negative bacteria like E. coli are different in that the cytoplasm is bounded by inner and outer membrane. Therefore any signal sequence will only result in the secretion to the periplasm. For those rare proteins that are secreted extracellularly from E. coli, an additional mechanism is needed to permit extracellular secretion, and this machinery is not mediated by the N-terminal signal sequence. So regardless of which signal sequence you use, in E. coli the protein will be periplasmic.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins