I understand that proteins with disulfides needed for biological activity/correct fold need to be expressed in the periplasm.
I wondered whether proteins without disulfides (cytosolic proteins) will endure negative effects when expressed to the periplasm (for example with pelB-leader-peptide in E. coli)?
So do I need two expression plasmids, one for periplasmic expression and one for cytosolic expression or is one for periplasmic expression sufficient for proteins with AND without disulfides?
Yes, you generally want to express each protein in the appropriate redox environment to either form its necessary disulfides (oxidizing, periplasmic), or to prevent it from forming inappropriate disulfides (reducing, cytoplasmic).
That doesn't mean you will automatically have problems if you don't do this right. But it will certainly improve your overall chances. Some proteins can cope. Others cannot.
Sometimes just expressing at lower temperature will allow the product to go to periplasmic space instead of formation of inclusion bodies. However inclusion bodies can be great for cheap protein purification - in some cases they can be solubilised and then refolded in a cystein-cysteamin buffer at 10oC.
Our group experienced E.coli expression with a thioredoxin-fusion protein vector, such as pET32a. Thioredoxin (TrxA) contains a non-canonical secretion signal and automatically secreted to periplasmic space. When using this system, we had 30~50% fusion proteins recovered from periplasm. It seems to be nice when expressing labile peptides, otherwise cytosolic proteases may break it down.
This may be written in somewhere of Novagen pET manual (I'm sorry, I cannot point the exact page now).
We experienced one very pity, technical problem upon using this secreted exprresion system. Usually, in order to recover the protein gently from the periplasmic space, we should use "osmotic shock protocol" for breaking E.coli outer membrane. This protocol is to be applied immediately after collecting E.coli cell pellets (without freezing), and may take a couple of hours or longer. This means I cannot go home after collecting cell pellets and keeping them in a freezer.
In our lab, still this TrxA fusion vector is the first choice to express 12 to 30 residues peptides. First we chose the standard method (freeze the cells, thaw, and sonicate). In some cases, not all, the peptide of interest is degraded and unable to purify by HPLC. Only in such the case, we use the osmotic shock protocol.
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