I expressed a cystein protease in M15 cells the vector is pQE30-xa. After lysis (8M urea in a tris buffer pH 8), the protein is in the pellet. I have tried many different things like 6M guanidinium hcl, sonication, microfluidizer, triton x-100, lyzozyme, and SDS. The only one that worked was SDS but since I need to do enzymatic assays SDS will interfere. What else can I do to bring my protein into solution?
Hi Marisol,
Have you tried expressing this protease at 20 degrees celcius? This might give the protein a chance to fold properly. If this does not work by itself, you should try combinding low temperature expression with using a standard lysis buffer containing lysozyme, DNase and 500 mM NaCl. If this does not work, express the protein, lyse the cells and centrifuge the lysate. At this point, the inclusion bodies are in the pellet so you can dispose of the supernatant containing all of the soluble cytosolic proteins. This provides a great purification step. Then you can solublize the inclusion bodies over night in 6M guanidine-HCL or 8M urea. The next day, the solublized inclusion bodies can be refolded by rapid dilution into a large volume of refolding buffer at 4 degrees with stirring. The solublized inclusion bodies should be added drop wise to the refolding buffer The solublized inclusion bodies might require centrifugation if aggregates are visible prior to rapid dilution. Also, the refolding buffer might require optimization. I hope this helps.
If it's not the case i suggest to refold it by flash dilution or dialysis.
You can do some TSA experiment to know the best buffer for the protein stability.
First question - why did you use urea for lysis? One only does this if one expects the protein to be purified to be insoluble.
Dod you? The first thins I suggest is to do a 'normal' lysis and see where the protein ends up.
I to suggest to first do normal purification even if its cystein protease, what is the range of protease activity?.
Thanks so much for your suggestions. Yes i did refold it. I used urea to solubilize it due to a protocol we have in lab for this enzyme. The thing is that this new enzyme has a very hydrophobic signal peptide added by mistake. Now i know my protein is making inclusion bodies. That is why sds is the only one to dissolve them. Now i need to find out how to get rid of sds because it interferes in enzymatic assay detection. What should i do??
For protein expression test, I would rather to use different cell host than only one cell host in small scale expression, like 10 mL. Play with combination of vector and host is also one way to make your time become efficient. Furthermore, as Lynne Regan suggestion, normal lysis is the simplest way to destroy the cell before we go further to any additional chemicals.
"The thing is that this new enzyme has a very hydrophobic signal peptide added by mistake. "
I may be missing something basic here but why not correct the "mistake" and get rid of the signal peptide? Seems like that may address the problem.
Hi Marisol,
Have you tried expressing this protease at 20 degrees celcius? This might give the protein a chance to fold properly. If this does not work by itself, you should try combinding low temperature expression with using a standard lysis buffer containing lysozyme, DNase and 500 mM NaCl. If this does not work, express the protein, lyse the cells and centrifuge the lysate. At this point, the inclusion bodies are in the pellet so you can dispose of the supernatant containing all of the soluble cytosolic proteins. This provides a great purification step. Then you can solublize the inclusion bodies over night in 6M guanidine-HCL or 8M urea. The next day, the solublized inclusion bodies can be refolded by rapid dilution into a large volume of refolding buffer at 4 degrees with stirring. The solublized inclusion bodies should be added drop wise to the refolding buffer The solublized inclusion bodies might require centrifugation if aggregates are visible prior to rapid dilution. Also, the refolding buffer might require optimization. I hope this helps.
Hi all,
Yes I was thinking to get rid of the signal peptide, but I was trying to avoid it. It may be the easiest way at this point. I will try Karen's suggestion first and if this does not work I will move on to sub clone my insert.
Thanks so much to all of you!! This forum is the best :)
I did not see you or anyone else mention it, so I will. If your protein has Cysteines, look at protocols that address this as well.
Hello Marisol! I'm trying to use the protocol that Karen suggested because I'm facing the same problem of having proteins in the pellet. Did it end up working for you ?
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry