mb at laplace.csb.yale.edu
Wed Aug 25 08:13:24 EST 1999
Rajesh Kumar wrote:
> I have expressed a new protein in BL-21 cells using pET-28 vector. I
> could purify it under denaturing (in presence of urea) condition using
> HIS tag. The problem is that as soon as I try to get rid of urea by
> dialysis (against PBS) or ultrafilteration the protein comes out of the
> solution. I tried to dissolve the precipitates in detergents (triton
> X-100, Tween-20), low pH buffer, high pH buffer but without success.
> On the basis of amino acid composition the protein (18 Kd) is slightly
> on the hydrophillic side except a 20 aa signal peptide region. Its
> predicted pI is ~ 9.7 (The pI of the protein itself is ~5.3 but the
> extra amino-acids that come from the vector make it 9.7).
> I will appreciate if somebody could suggest me a way to make the
> protein soluble in a system that doesn't interfere in most protein
> analysis work.
> rk6n at virginia.edu
> University of Virginia, Charlottesville .
I'm a little unclear on what you are attempting but I'll give you a few
things to consider. First, purifying proteins under denaturing conditions
is generally a method of last resort as many proteins will not refold
easily once denatured. You may want to look into how you are removing the
urea. You could try doing this at lower protein concentration or utilize a
rapid dilution method rather than dialysis.
Second, the signal sequence is usually analogous to a transmembrane
segment. These are generally cleaved from the final protein. If you don't
need it, I'd reclone the protein without it. This may make eliminate the
need for denaturing purification altogether. If you do need to keep it,
I'd suggest treating this like a type I or type II membrane protein and
including some mild detergent during the purification. Once the protein
precipitates, these mild detergents will not be very effective. I use
Thesit because it is inexpensive and doesn't have UV absorbance.
Dept. of Molecular Biophysics and Biochemistry
More information about the Methods