I am having a problem with a protein I recently purified on a His
column. Some of this may be basic, because I'm an immunologist and not
very experienced with protein purification!
I am trying to isolate SDF-1a (CXCL12) from a bacterial expression
system in BL21-DE3. I have gone through a basic optimization for
expression, and can detect protein in my lysate. The protein is 6
His-tagged. The column is Novagen's His-Bind column.
The cells were lyzed in 20mM Tris-HCl pH 7.9, 5mM imidazole, 0.5M NaCl,
1% triton X-100, 1mg/ml lysozyme + protease inhibitors. Elution was in
20mM Tris-HCl pH 7.9, 0.5M NaCl, and 1M imidizole. The protein came
off in Fraction 2, and was (apparently) soluble at that point.
Analysis by anti-SDF1 WB, coomassie, and ponceau show only the single
band, so it looks pretty pure.
I then dialyzed against PBS overnight. I need to do an N-terminal
coupling with this protein, and the Tris will interfere. During the
dialysis, the protein came out of solution. I have checked the PPT and
supernatant, and essentially all the protein is in the ppt. I have
tried to get the ppt to resuspend in dH20, and PBS with 0.5M NaCl to no
I have checked the pI of the protein, and it is reported as 9.5, so I
am well away from that. The readdition of Triton x-100 also did not
seem to help solubility. Does anyone have any suggestions? I need to
keep the protein biologically active, although it may be that the ppt
is a sign it has denatured and lost activity already. But I used only
about 1/4 of my lysate, so any suggestions could be applied to later
Any suggestions would be most, most welcome.
University of Colorado HSC/National Jewish MRC