I want to express a protein which comes from plants in E.coli for assays and structural studies. I am ordering a synthetic version of the gene that is codon optimized for E.coli. When I run the sequence through multiple prediction servers, there appears to be a transit peptide sequence that gets cleaved as the protein enters the chloroplast.
Should I just trust these prediction servers and order the gene with the predicted targeting sequence removed? Or is there a way to cleave the target sequence later...using an enzyme?
I do not have experience, but I do have in mind, that target sequences often have negative effect on the native function, if not cleaved.
A professor told me, if I would ever will get in contact to this problem, first try it without the target sequence.
Cleaving it later, adds one more experiment to your pipeline, that can fail (including setting up the experiment, find right solutions and ph, temperature, ...). Besides the valuable time, you also might prepare to spend more money.
I agree with Jorg - the signal sequence is very likely to give you problems. A critical reason for this is that the signal sequence is usually designed to act as a transmembrane helix, and so is likely to be very hydrophobic. This can often make your protein a lot less soluble.
If you think that your protein might prefer to be expressed by being passed through a membrane, then it would be better to try and get the protein sent to the E. coli periplasm. There are good vectors available (e.g. pET26b) that include the E. coli signal sequence, which will result in the protein being passed to the periplasm. Of course, you can get this engineered onto your synthetic gene if you want. In general, one tends to get lower levels of expression in the periplasm (as E. coli can only pass so much protein through the Sec pathway), but for some proteins, it makes all the difference.
Good luck!
Thanks for the replies. I am working on this now and will share with everyone how things go.
Update: I have successfully expressed and purified the protein using E.coli. Today, I begin protein crystallization trials.
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