Hello everyone, I am working with a difficult-to-express proteins and have changed expression host and cloning strategies a number of times. Finally, I was able to get something with cloning in pET28a+ EcoRI/Xho I sites. Unfortunately, all my protein is found in the pellet (I induced with 0.5mM IPTG at 30°C) . Someone suggested that it may be because, I did not remove the 30 aa signal peptide residues. How does this affect protein expression? Also, my protein is 279 aas with 8 Cys residues. How do I optimize this to have more soluble protein? How do I go about with the solubilisation and refolding?
The first thing you're going to want to know, is if these cysteines need to be reduced or oxidized. For intracellular E. coli expression, naturally intracellular proteins with reduced cysteines are certainly much better suited.
I'm guessing the reason your colleagues think it's difficult because of the cysteines, is that they're supposed to be oxidized. That would be so for example, if your protein is from a eukaryotic host and is naturally extracellular. This is one of the things E. coli is notoriously bad at. Specifically, once the proteins exit the reducing intracellular environment, the cysteines might oxidize randomly, causing them to pair incorrectly.
If that's what your problem is, it could be arbitrarily difficult to solve. You could try to use chemical re-folding using a "redox buffer" system, perhaps one found in a commercial "re-folding condition screening kit". Or you could aim for soluble expression using a fusion protein strategy. For example the MBP fusion system is excellent at promoting solubility of otherwise intractable proteins. (if it is a protein with naturally reduced cysteines, MBP fusion alone would be likely to solve your problem).
But ultimately, if the cysteines are naturally oxidized, it's entirely possible that a highly yielding solution for this kind of problem cannot be found in E. coli. In that case, it'd be safest to express it in the organism or cell culture that is closest to its natural host.
For expression of soluble, disulfide-bonded proteins in E. coli, you could try the SHuffle strain from NEB, which has been engineered for this purpose.
https://www.neb.com/applications/protein-expression-and-purification/expression-of-difficult-proteins/disulfide-bonded-protein-expression/methods-of-disulfide-bonded-protein-expression
As for the signal peptide, if it is not needed as a secretion signal, you should remove it from the construct because it is hydrophobic.
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