[Protein-analysis] Protein Solubility Problem

rice.jeffrey at gmail.com rice.jeffrey at gmail.com
Fri Aug 11 10:44:37 EST 2006


Hi,
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
avail.

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
runs.

Any suggestions would be most, most welcome.

Thank you,

Jeffrey Rice


University of Colorado HSC/National Jewish MRC
Denver, CO



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