I'm trying to solubilize a protein using TaKaRa chaperone plasmids. I use IPTG and Tetracycline/arabinose for inducing my protein and chaperone plasmid respectively. Either my protein gets expressed or the chaperones, but never both. Please suggest me some troubleshooting steps. Thank you.
There are cases in which chaperone overproduction simply accelerates degradation/proteolysis of the target protein (see e.g. PMID 20813055; there is a relatively recent review of this topic somewhere in the literature but I can't seem to find it now). Or, there might simply be some experimental error you are unaware of. If you have a second, non problematic protein in the same vector I would use that as a positive control.
Is the expressed protein soluble/useful? It does not matter if the chaperone is expressed or not. If it is not soluble (I assume that is the case) here are a few things that you can think of:
1.How are you looking for the soluble protein? Can you pick it up using a western blot? The SDS-PAGE comassie may not show a good thick band.
2. Do you have an assay for this protein? (is it an enzyme?)
3. See PMID:19074766 for a test case. The takara chaperone system works only sporadically and many proteins are still unstable and the increase in yield is minimal. Make sure your lysis buffer has a variety of protease inhibitors.
4. Do a careful time vs inducer (Tet/arabinose, or whatever) profile at various cell densities(0.5, 0.8 and 1.2 OD600). If you are lucky there might be a condition that is optimal but not by much. This may be good enough for your purpose. Induction temperature is another variable that you might add to the mix. It is quite a bit of work right there.
Good luck
Thank you very much Alejandro and Sujay. We don't have a positive control and after rectifying the mistakes with O.D, now we could standardize the concentrations and O.D and have got a good induction following your suggestions. Now on our way to induce both of them together. Hoping to get good results soon. Sujay, as your 1,2,3, suggestions were not applicable to our protein and I followed 4th suggestion which is working good.
Thank you very much,
Kind regards,
Manjusha Pai.
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