Does SUMO fusion protein increase solubility? - (Jul/11/2005 )
I am doing overexpression of my protein, but whenever I overexpress the protein, I only have inclusion body. I tired different temperature (25C, 16C) and different IPTG (25uM to 500uM), but it does not help at all. Even I found that my protein is all inclusion body without IPTG induction. Im addition to that, N-terminal is very critical for protein activity. Some people suggest that SUMO fusion protein increases solubility and gives navie form. But, I am not sure whether it can increase the solubility for prokaryotic protein.. So, if there is someone has some exprience with this system, could you give me some suggestion, please? It has been almost 7 months to have this problem and I am very sad!!!!
Thank you all.
I'm sorry to come back to your problem of almost two years ago,
but I just saw it and I was wondering whether you actually tried the SUMOsystem?
I'm considering to use SUMO-fusion-expression myself,
but since there are so many other systems out there as well
it's hard to make a decision what to use!
Hi Micha and Menita,
This is a hard question to answer because every protein is different but I will tell you about my experience with SUMO. Me and one of my labmates have tried SUMO to purify various proteins from Streptococcus and our experience with SUMO has not been the best. After cloning and overexpressing my protein half of it was soluble half of it is not but it was enough to purify. I purified with out any problems but I had the hardest time cutting the SUMO tag of my protein. After you purify your protein you have to dialyze against PBS or Tris-glycerol buffer in order to cut with the protease, about 80% of my protein precipitated in the PBS and by playing with the salt cocnentration I was able to recuperate some protein in the Tris-glycerol buffer. Ayways after you cut the protein you have to separate the tag from your cut protein and my protein even though it was cut was binding non specifically to the resin in the nickel column. The thing was that even if I started with 2L of culture I ended up with like 500ng of protein which is not enough for my stuff. My labmates experience has been that one of the proteins she tried to purify was still in inclusion bodies even with the tag and she tried a bunch of detergents and nothing work. Her other protein previously with a His tag it was soluble and with the SUMO it was half soluble and half in inclusion bodies. After she purified this protein and cut the tag she tested the protein and it was inactive with out the tag and active with the tag.
Talking to other people in our department most people have problems cutting out the tag and troubleshooting conditions for the protease. If you can afford to keep the tag on for your future experiments I think it may be worth a try but don't get your hopes too high for SUMO because it is not as great as people make it sound. Whatever you do do not buy it from Lifesensors their technical service is awful.
I hope this helped in some way.