Hi everyone
While working with a protein in P3E construct, the protein seems soluble in presence of CHAPs in lysis buffer (also screened Tween 20, triton 100, DDM) detergent, i purified it using GST column. But after dialysis (in which the CHAPs was removed) and incubation with precission oovernight at 4*C,it failed to cleave of the tag.
I was expecting a cut protein but the intensity of uncut protein is more then the cut. Also i used 1:5 ratio of precission protease.
Since GST is intact and worked effectively for 1st column, i don't understand why my protein acted as if it mis-fold.
I tried running a SE (without CHAps) as well and all my protein is in void peak.
With the void collected smple i retested the cleavage by adding CHAPs 0.1% and precission again, but after 8 hrs of incubation also the uncut protein still remains.
Is there any way to recover the protein? Or any suggestions to improve the purification methodology?
thank you
Here is my hypothesis. The protein may be insoluble without CHAPS or some other detergent. When the detergent was removed by dialysis, the protein may have aggregated, but not visibly precipitated thanks to the GST tag and residual CHAPS. The cleavage site for the protease became inaccessible in the aggregate. The high molecular weight of the aggregate resulted in its coming out of the size exclusion column in the void volume. The solution may be to keep the protein in CHAPS, or other detergent, throughout the procedure.
Dear Adam
Thank you so much for your valuable suggestion.
In recent trial I tried 6M Urea for denaturing and then dialysis (2Lx2)to remove urea. And then added precission protease, kept for 3 hrs,but it still remains uncut.
So, for next, i'll keep it along with CHAPs in all steps.
Thanks a ton.
:)
How about to cut first and then dialyze?
If you write you see more uncut than cut, how did you test that? By SDS-PAGE? In that case it shouldn't matter whether it precipitated or not (if you analyze it directly from the cleavage reaction, without spinning). Thus I'd expect problem with the cleavage. Can you test with different protease (from different lot, or borrow from some other department?) or with other protein, that you know it works.
If it will not work with other protease and/or work with other protein, I'd suspect some problem with the cleavage. How long is the link between GST and your POI?
Did you try on-column cleaving?
How about to denature your protein, remove urea (in one step), cleave and re-nature (add urea and then slowly remove)?
Question- do include a reductant in your cleavage buffer? Precission (HRV 3C protease) is a cysteine protease and requires a reductant (BME, DTT, GSH etc.) to be active.
Dear Micah
Yes i add 2mM of BME in all buffers and also in dialysis buffer.
Dear Tomas
There is GST then 2 A.A followed by precission site then 4 A.A and then POI.
We express our own precission and it worked for my other labmates so hopefully it is working fine.
I am planning to try co-expression with some interacting partner of my POI. Fingers crossed.
only so few? That could be a problem, that it's hidden and the protease cannot reach it.
Did you try exactly the same batch? The same tube of the protease? Or it work in the past?
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