protein insoluble in just about everything! - (Sep/06/2006 )

Hi,

I am trying to purify a membrane associated his-tagged recombinant protein from E. coli culture.

I originally tried a range of different detergents (CHAPS, cholate, n-OG, DDM, Triton X-100) and they did not solubilise the protein at all. I then assumed my protein was in an inclusion body, as there were huge quantities and the pellet was remarkably pure.

I then tried to resuspend in DMSO - protein concn in the supernatant after doing this was so low, that I could not run on an SDS-PAGE gel to see whether or not my protein had been solubilised at all.

I then tried under denaturing conditions (8M urea). Most of the protein remained in the cell pellet, although a relatively small amount was solubilised and eluted at pH 4.5. When I dialysed this however, the protein precipitated out of solution again! I tried increasing volumes of my buffer to assist in solubility but this did not work.

Any other suggestions? I am running out of ideas. I thought about using guanidine HCl, but am really running out of time for my project, as my lab work stops in a month

Please help me!

Also, one more thing... Is it strange that none of my protein was found in the supernatant at all? I thought that some of it would have been folded correctly before the aggregation occurred...

Hi,

I am trying to purify a membrane associated his-tagged recombinant protein from E. coli culture.

I originally tried a range of different detergents (CHAPS, cholate, n-OG, DDM, Triton X-100) and they did not solubilise the protein at all. I then assumed my protein was in an inclusion body, as there were huge quantities and the pellet was remarkably pure.

I then tried to resuspend in DMSO - protein concn in the supernatant after doing this was so low, that I could not run on an SDS-PAGE gel to see whether or not my protein had been solubilised at all.

I then tried under denaturing conditions (8M urea). Most of the protein remained in the cell pellet, although a relatively small amount was solubilised and eluted at pH 4.5. When I dialysed this however, the protein precipitated out of solution again! I tried increasing volumes of my buffer to assist in solubility but this did not work.

Any other suggestions? I am running out of ideas. I thought about using guanidine HCl, but am really running out of time for my project, as my lab work stops in a month

Please help me!

Also, one more thing... Is it strange that none of my protein was found in the supernatant at all? I thought that some of it would have been folded correctly before the aggregation occurred...

I have a similar-sounding protein. 100% in inclusion bodies even when induced at 22 degrees.

Apparently refolding on-column often works well because the protein is immobilised at one end and can't form aggregates. I am trying dialysis of my resin: I adsorbed the protein onto my nickel resin in 8M urea, put the resin in a dialysis bag and dialyse against 4M, 2M, 1M and 500mM urea with 10% glycerol (24 hours each). Eventually I hope to elute with imidazole with a little triton-X. I don't know yet if this will work, but it's an idea.

Did you sonicate while you used detergents?
I use PBS plus 1% triton X-100 and I sonicate, unless I'm not able to solubilise the protein I'm interested in.