working on a series of insoluble recombinant proteins with a 6xHIS tag (N-term). Solubility analysis has been done and pellet MUST be dissolved in 6M GuHCL.
Following pellet re-suspension for one hour, it is applied to Ni-NTA and subsequently washed in a buffer series containing 20mM Tris-HCl/250mM NaCl/6M GuHCl/1mM Beta-Mercaptoethanol and increasing concentrations of imidazole ranging from 5mM to 500mM.
Protein has found to elute at 20mM Imidizole.
There is the first issue - SEEMS like a low affinity to Ni-NTA.
FYI - The isoelectric point of the protein is 5.7ish and buffers are at 7.5-8.0
Then there is the next and MUCH bigger issue.
Proteins are dialyzed against 20mM Tris, 150mM NaCl, 0.3M Arginie overnight and upon coming in in the morning there is a pretty good amount of precipitate right in the dialysis casette. Spun it down and looked at the supernatant and the precipitate pellet on SDS-PAGE and yep, the protein is in the precipitate.
Now one big thing is that this is a protein fragment so I'm not sure that re-folding is a huge issue.
What I am in the middle of right now is that I loaded the 6M GuHCl dissolved pellet onto Ni-NTA for an hour. Then, ON COLUMN I am running a 6M to 0M UREA gradient (in 20mM Tris,250 NaCl, 5mM Imidazole + Urea) and THEN doing my washes and imidazole elutions without GuHCl present. Going to run those out on a gel soon and see if it still crashed on column.
So if anyone has any ideas about the low affinity and more importantly if anyone knows of a magic buffer I can dialyze in that would be so appreciated. I have to try and keep it somewhat physiological, detergents are ok. I many nay people have had this issue when you try to get things into a physiological buffer from Urea or GuHCl.
Speaking of, I have also tried a TBS +0.5% Tween with a hint of EDTA (say .5mM) dialysis buffer and not good. This buffer was used afer column elution.
Also, I am about to try tonight...Dilluting the protein out to a very low concentration , I have no idea what it is now but still, and do a dialysis with that. I can only say that after running it out on a gel there is a good deal of protein in there so possibly dillution might help.
I know 6M GuHCl is common on Ni-NTA so I don't know. I would like to do a series of micro-dialysis screens but we have neither the supplies or time at this point.