Deal all, thank you for all the wonderful helping tips. A quick test
of ATP-Mg at 2mM at 37C for 2 hours had proved ineffective in my case
so now I'm thinking of trying the other protocols that you had offered
as listed below. Many thanks and have a wonderful week! -yong

Original Question
RE: Removal of bacterial chaperone Hsp70 contaminant from recombinant
protein preparation

Dear all,

I have a protein expressed at 37C for 3 hours in BL21 DE3 and purified
with sub-stoichiometric amount of apparent Hsp70 contaminant even
after exhaustive affinity (GST-fusion or His-tagged), ion-exchange and
sizing column steps. I would like to know if you have a
well-established protocol for getting rid of such a contaminant. I
asked around and was told to try adding ATP at certain(?) stage of the
purification.

I would much appreciate your input.

Many thanks! –yong @ the Wistar Institute

Response 1:
Try 1-3M urea in the lysis buffer, and maybe first wash buffer. If you
use it in the wash buffer add some lysine (maybe 20mM) to prevent
carbamylation of your protein.
Kendall

Response 2:
Hi Yong,
I add ATP to my purification.  If binding to Nickel column, I would
wash with ~10 to20 mM imidazole, then wash with the same wash buffer
PLUS 1-2 mM ATP, then rewash the column to get rid of any residual
ATP.  You can then elute your protein and hopefully be rid of the
pesky Hsp70.
Good luck,
Jill

Response 3:
I routinely add Mgcl2 (0.5 to 1 mM) in my all buffers and also after
purifying protein from Ni-NTA, I incubate it with 1 mM Mgcl2 and 1 mM
ATP at 37 C temp for 15-30 minute followed by second step of
purification.
This way I get rid of HSP contamination....
Hope this will help
Radha

Response 4:
We managed to reduce, but I don't think absolutely completely remove, chaperone
contamination by putting ATP in a column wash buffer (while the
protein was bound).  The idea was that ATP hydrolysis should make the
chaperones let go of your protein.  The level remaining was only a
problem for doing ATPase assays of our protein, which should be nearly
inactive in the absence of DNA - even essentially coomassie-invisible
levels of chaperone messed up the no-DNA controls. I think my troops
also tried adding purposefully denatured goop to the prep to lure away
the chaperones, but decided that just complicated things.
Phoebe

Response 5:
I had similar experience once and filling the column with 5 mM Mg-ATP solution
and living it like that for few hours, followed by good ATP wash (few
more column volumes of Mg-ATP wash), helped a lot.
YMMV.
Dima

Response 6:
Have a look at the following papers
Proc. Natl. Acad. Sci. (1995), 92, 1948.
J. Biol Chem (1984), 259, 8820.
Gabriel

Response 7:
I used Tris 10mM, MgCl2 10mM, ATP 5mM (adjust to pH=8.5, w/o buffer,
it would be very difficult to adjust the pH value) to wash the resin
(10-20 column volume) during on-column wash, and it works.
Yeming Wang, Ph.D.

Response 8:
Hsp70 is very hydrophobic, and the problem mentioned by you is quite
common in many affinity-based purifications.  I suggest that you load your
sample on an HIC (say, phenyl sepharose) and elute with a gradient from
high-salt to no-salt.  Hsp70 typically elutes close to no-salt buffers on
the HIC chromatography.
Shekhar Mande

Response 9:
If you are trying to purify away a chaperone protein, I found the
following protocol useful: Chen et al. (2001) J Mol Biol.
307(1):173-82
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11243812
However, you should also consider the following:  If you really can't
get rid of HSP70 - even after extensive purification effors - this
probably means that the chaperone is binding to your protein (Probably
because your protein is not correctly folded, e.g. it may have
surface-exposed hydrophobic patches). In this case, even it you manage
to "knock off" HSP70 contaminant from your protein, you protein will
probably aggregate and/or precipitate. Good luck,
Chris

Response 10:
Perhaps try one of these:
1. Wash with ATP+Mg when bound on the lMAC column (to get it to
release your protein).
2. Wash with 0.1% Triton when bound on the IMAC column.
3. Hydrophobic interaction chromatography.
4. Wash with 0.5 GuHCl while bound on the IMAC column (pray!!)

On a sad note, the protein will quite often aggregate when it is
released. There is a reason why the only sign of target protein you
see is stuck in a chaperone... but at least you have the chance to
scout around for a good buffer to release it into.
Martin

Response 11:
add 10 mM ATP + 2.5 mM MgCl2 in your purification buffer and wash it
off during your affinity step...this has worked for me (of course make
sure that you're not unlucky and your protein doesn't elute during
this step; don't worry it shouldn't) if that doesn't work, maybe try
changing some things on the cell growth and expression end, like
induction at lower temperature or a different BL21 strain like pLysS
or STAR
HTH
Brad