Hi,
I am working with a protein that is typically secreted from the parasitic cells that I study. I am trying to express and purify it from HEK cells. Despite of using the mammalian signal peptide, it is not being synthesized as a secreted protein. I was wondering, if expressing the protein as cytosolic in the HEK cells (by removing the signal peptide sequence) would interfere with the folding of it? We plan to do activity assay with the purified protein. So proper conformation is necessary.
Thanks for your suggestions.
Sumit
Dear Sumit
i do not think that citosolic expression will affect the protein folding but it will be certanilly make more difficult the purification. Moreover i suspect that no secretion in Hek293 can be related to the unfolding of the protein that aggregate and cannot be secreted from the cells, also if may depend also from the intrinsic properties of the protein. Gfp for example is a good control for citosolic expression but not for secretion in hek293 but the fact that your protein is tipically secreted make me doubts about its correct conformation. Did you express the full lenght protein or a domain?
Which signal peptide sequence did you use?
good luck
ciao
Manuele
Greetings,
This is a question that could deserve a dissertation, but for this forum a good answer will depend largely on your protein's (1) post-translational modifications (N- or O-glycosylation, deliberate partial digestion as with prohormones, disulfides, etc.) and (2) dependence on specific chaperones (for folding, secretion, or both). If the heterologous system well duplicates the mother system in these ways, then the odds of a stable, abundant native product are greater.
Another challenge may be whether the final product is homogenously well folded or whether you possess plural populations of conformers, only one/some of which is correct. Obviously, you would dislike this as well. Does your field have a fair grasp of this protein's "free energy landscape" ("folding funnel"), as understanding its transition states can inform whether this is to be ignored?
As we often see, there are countless details that *could* plausibly fail. For a secretory protein expressed in cytoplasm, for instance, if your protein favors a specific salt, protein-protein or pH conditions not supported in this environment, its nascent conformations may get stuck in some variety of kinetic trap prior to arriving in the native state and such a delay could lead its folding off-track, promoting aberrant shapes or aggregation.
To be clear, my belief is that these final two paragraphs should not concern you as much as the matter of post-translational modification compatibility between expression systems. That's also the concern that is easiest to predict based on the literature, whereas the others are sneakier issues that you'll probably only learn after trying.
All the best,
Dave
Hi there,
If your protein is able to fold properly on its own there is no problem. As mentioned above the issues are linked to the natural protein processing (post translational modifications) and the possible presence of disulfide bridges in the native protein. If both are necessary for protein biological activity, your cytosolic expressed protein would be inactive and structurally unstable although it might be properly folded at once.
Depends on the protein. If the protein has disulfide bonds, cytosolic expression is not a good idea. Same thing if it depends on interaction with resident chaperones in the secretory pathway or if produced as a propeptide that must be cleaved somewhere along the secretory pathway. Some proteins do not fold well if not glycosylated, those would also be poor candidates for cytosolic expression.
Hope it helps!
You say "parasitic cells". Is your parasite a eukaryote? If so, a secreted protein goes through the ER and is glycosylated there; -SH-groups are converted to -S-S-. Cytosolic expression will then likely fail.
As the previous comments mentioned, proper folding depends on the character of your target protein. I think the safest way is to add the appropriate secretory signal peptide to your protein of interest.
Good luck!
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