1 reply to this topic

[teka]

Hi all,

I work with a 50KDa his tagged protein (cloned in pET 28 and pET Sumo) that is overexpressed in Rosetta cells. I have a solubility  and purification problem.The protein is only soluble (partially) in the presence of 0.3% of Sarkosyl (I've tried different concentrations of IPTG, different temperatures, and different pHs in the lysis buffer). However, with sarkosyl the protein does not bind to Ni-NTA. I've tried to dialyze the lysate but it didnt work. I' ve heard  about adding triton and chaps to the lysis buffer to make the protein bind to the colunm in the presence of Sarkosyl. The protein bound indeed, but then it doesnt come out, not even with the addition of EDTA! I already tried to express the protein in pMAL vector, but I had the same problem.

I'm expressing the protein with 1mM IPTG for 3 hours at 37C. My lysis buffer: 50mM Tris-HCl pH 7, 500mM NaCl,0.3% Sarkosyl and protease inhibtors, then I sonicate the cells.

Any of you gurs have an idea of what can I do to purify the protein? I will need the protein for crystallization assays.

Thanks in advance!

Teka

[mdfenko]

is the native protein insoluble or only the expressed protein?

you can solubilize by denaturing with urea, purify with ni-nta then refold by dialysis.