Protein Solubility

Rajesh Kumar rk6n at virginia.edu
Sun Aug 22 18:55:43 EST 1999


Hi,

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.

Thanks

Rajesh

rk6n at virginia.edu
University of Virginia, Charlottesville .




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