I've faced a little problem. I used E. coli to express an archaeal protein that should be thermostable. When I used heat-treatment to remove the bulk of the endogenous E. coli protein for purifying my thermostable target protein, my target protein is also lost during the heat-treatment step. I am pretty sure that protein should be thermostable.
1) Have you modified your protein of interest in ANY way (truncation, tags, etc...)? Such modifications may change its localization / topology =>localization.
2) How about the promoter? Bulk proteins over-expressed under constitutive / inducive may be deposited elsewhere than predicted (inclusion bodies; self-segregates, etc.).
3) Maybe it'd be good idea, to use an archean host for o/x as well...?
These are kind of general quesions, which one should pose designing such experiments :)
there may be the problem in proper folding of that particular protein in E. coli
Hi William! I think you haven’t spotted well the source of your problem. Several labs expresse Taq pol in E.coli and I can tell you it is just fine. They even purify it by just boiling the whole stuffs. We have done it in one of my previous labs. So, maybe you should look for other factors. Did you check for the codon usage frequencies between the two species? You should optimize this parameter first. Even if your protein is toxic or less soluble you may still be able to produce it. Report back! Good luck!
We did face the same problem when purifying the SSV1-integrase from E. coli. However, when partially purified, it was possible to perform heat denaturation without loosing the target protein. My hypothesis was that in a crude extract concentrated around 15 mg/ml denatured proteins would act as "carrier" if a little part of the expressed protein is disordered. Over expression of E. coli chaperons may help (that was not the case for Int-SSV).
To start with, you can probably run an electrophoresis of the mixture of proteins, and compare it with the original archaeal protein, just to see if there are any structural or compositional changes. If there are any, you can use any other method for separation.
Hi william! I thought you didn’t expresse it at all. How do you know that the denatured protein is yours? Have you performed MS analysis? As suggested above you can co-expresse E. coli Hsp 70 as suggested by Marie. You can also try to refold it. There are a lot of protocol which work. You can solubilize your protein in 8 M urea buffer and then dialyze it against decreasing concentration of urea until you reach 0 M. Normally I would expect that a heat-resistant protein will refold quite nicely.
Report back how you deal with the issue.
If you are detecting activity prior to the heat treatment step and no activity after, then you may be losing your protein due to proteolysis. Are you adding protease inhibitors to your cell extract prior to the heat treatment? If there are active proteases in your extract and your protein has an amino acid sequence that is sensitive to proteolysis, as you raise the temperature, the activity of the protease increases (up to a point) as well as the likelihood that your enzyme will be attacked and inactivated. At some point the activity of the protease will also suffer due to the increase in temperature, but the damage may already be done. Just my 2 cents worth.
answer for Abdelhalim Boukaba : Taq polymerase is not a good comparison on this specific problem : it has a bacterial origin (Thermus aquaticus) not an archaeal origin. From my own experience that makes a big difference at each step of expression/purification of thermophilic recombinant proteins in E. coli.
Dudley Eirich raised an important point, protease inhibitor coktails should be used (eventhough raising the temperature oftenly help thermophilic archaeal proteins to fold tightly, hence been less sensitive to proteolysis)
Thanks Marie! I don’t have any experience with expressing proteins from Archaea in E.coli. I take your suggestions on that because of your expertise in this matter. I always assume that everyone trying to purify whatever protein from whatever specie include protease inhibitor cocktail in his buffers during all the purification steps.
So, I think the systematic way to proceed is to confirm first that there is expression of the protein and then proceed. If it is misfolding then one should try to find the way to turn around. Does it happen before or after extraction? If William starts heat-treating E.coli cells, then there is no way to know that.
I found the answer. Buffer is the most critical to my target protein.
pH and pHi are often the key for protein solubility ! Great to know that you made progresses on your purification
Hello... I have a logical answer. consider this. Since u are cloning a thermophilic protein in E.coli and then purifying it.. there are possibilities for its homolog to be present in the crude lysate. if ur protein has the tendency to oligo merise the treatment at 10 min or 20 min might show fainter bands because of hetero oligomerisation of ur protein with the archael protein. try treating for say 1 hr or so. if u still dont find a strong band then i dont know what to say... But this might be a logical guess...
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