HSP70 contamination after Nickel purification - (Jul/09/2016 )

Hi All, 

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.  

Thanks. 

Some options to consider:

1. Use higher salt concentrations during your nickel wash, which may disrupt interactions with HSP70 (e.g. 0.5-1 M NaCl)

2. Small amount of mild detergent may also help accomplish the same thing (e.g. 0.1 - 1% Triton X-100)

3. Washing with a mild denaturant/chaotropic agent like 1-3 M potassium thiocyanate, then wash away chaotropic agent, then elute off resin.

4. Completely denaturing your protein in 6M guanidine or 8M urea will certainly do the trick...but requires that your protein also refold properly. Perhaps try loading your protein onto the nickel as you do, then do an 6M guanidine wash...then wash with 4M, then 2M, then 1M, then finally back to your original wash buffer to refold your protein. Then elute as usual. If you have an FPLC, you can do the re-folding with a slow linear gradient from 6M to 0M guanidine while your protein is on the column. Note that 8M urea crashes out at 4 degrees C.

5. Try expressing a different ortholog of your protein. Sometimes one ortholog will stick to all kinds of garbage, express poorly, etc...but it's relative with 60-90% sequence identify and functionally identical will express and purify like a dream. 

Thanks for the reply labtastic. 

1) My wash buffer already has 500mM NaCl, but I can increase it to 1M NaCl and see if that helps. 

2) I'll try adding some detergent.

3 and 4) I worry about adding denaturants as I don't want to denature my fragments as they are all ready quite big and I don't want to get an unfolded proteins. 

5) The only other ortholog of my protein would be flies, I'm working on a C. elegans' protein. After flies would be human, but then there was a duplication event that gave two orthologs. This sequence identity isn't ideal as well with only 37% of the sequence being identically (which is really good for a worm protein). 

When you was with ATP to release HSP70, do you also have magnesium and potassium salts in there as well?

http://www.ncbi.nlm.nih.gov/pubmed/8413631