My fusion protein has MBP and small peptide (~5kDa in monomeric form and 25kDa in pentameric form). In between my fusion protein, I have cleavage sequence for Factor Xa protease and TEV protease. My fusion protein is in phosphate buffer. I have tried Factor Xa but it doesn't seem to work; it also has lower specificity in comparison to TEV. With TEV, I am barely getting 5% cleavage for my fusion protein. I think my cleavage sequence seems to be buried. So far I have tried to add detergents such as Urea, SDS, Triton X-100, and Tween 20; and I left it on benchtop with TEV overnight at 20 (deg Celcius). Treating with urea seems to work a little bit. From looking at the Page gel my cleavage improved by 5% percent by treating it with urea. At this point I don't know what I should do? Any ideas, suggestions and experiences greatly appreciated.
Could it be that your target protein concentration is relatively high? Or is the TEV used not active enough?
All the best!
I have the same problem. I was told to try using another tag instead of MBP, for exemple SUMO.
Now, another possibility is that your protein IS cleaved but that your peptide just stays attached to MBP (typical of small TM proteins apparently) so you don't see it on gel.
Good luck !
Nish Rathod Hi, thanks for your reply. Can you please share how were you able to troubleshoot this? I am facing a similar problem. I have the TEV cleavage sequence in the middle of the protein sequence possible hindered, and so TEV enzyme isn't able to recognize and cleave it. Thanks!
I tried different buffers until I found one that worked. E.g., Tris, phosphate, etc.
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