Unstable protein - (Jan/13/2009 )
I have a His-tagged protein (with several cystein residues) that is insoluble after expression in E.coli. I can solubilize it in 8M Urea and purify it under denaturing conditions over Ni-NTA in batch. Then I dilute the eluate (which is in 250mM Imidazol and 8M Urea) 1:10 in 20mM Tris pH8, 0,5M NaCl and incubate again with Ni-NTA. This time I do normal native purification, elution with 250mM Imidazol, but without Urea. When I check the native elution fractions on SDS Page directly after purification they look good (quite a lot of protein), but after only few days (no matter if at +4°, -20° or -80°) there is hardly any protein left. Needless to say that after buffer change with centrifugal filter device (to remove Imidazol) I have no more protein at all.
By the way, the protein eluate fractions containing 8M Urea are stable at all three temperatures.
What can I do to get my native, stable protein ?
Please share your experiences.
your protein may not have refolded properly and precipitated (hence the loss when centrifuged).
you can try refolding by stepwise dialysis (slow removal of urea) to obtain proper folding (hopefully).