I am currently trying to express fragments of my full length protein, since the full length protein does not express well in e. coli.
One of my fragments is 540 amino acids (63kDa), one is 495 amino acids (58 kDa), and the last one is 213 amino acids (26 kDa). These are all C-terminal fragments, each with a 6X His on the C-terminus.
I am able to get them solubly expressed (something I was not able to get with the full length protein as after lysis and spinning down, it went into the pellet). However after Nickel purification, there is a huge band of HSP70 in the elutions for all my fragments. I tried doing a ATP wash, but the HSP70 is still there.
My current growing conditions are, I grow 1L TB (with TB salts) with 10mLs of overnight culture. I grow the 1L flask at 37*C at 250rpm till I hit OD 0.3, then I drop the temperature to 16*C and grow for 1hour. The OD at this point is usually 0.6, and this is when I induce with 10uM IPTG and grow overnight and collect the next day and freeze in liquid nitrogen and store at -80*C.
To lyse my cells I thaw the frozen pellets, add my buffer which contains Potassium Phosphate pH 7.45, 10% glycerol, 10mM Imidizole and 150mM NaCl, and lysozyme and protease inhibitors. I then sonicate to disrupt the cells.
So my questions is, is there any way to prevent the HSP70 from being expressed? Or am I just screwed.