Stabilizing overexpressed proteins

Emir ekhatipo at NOSPAMmidway.uchicago.edu
Fri Feb 9 20:23:39 EST 2001


I actually had problems with concentrating the pure protein, too. It would
precipitate at the concentration above ~5 mg/ml if dialyzed against 50%
glycerol in the same buffer (50mM HEPES [can be replaced with Tris or Pi],
200mM salt, etc.). I need the protein concentrated enough for further
crystallization. The latter limits the number of compounds I would like to
introduce into the buffer. High salt seems OK, same possibly with ethylene
glycol (since it by itself can be used as a crystallization agent), but what
about mild detergents (btw, thanks for reminding about good old cholate) and
betaine? Would not they be a problem during crystallization? It is quite
troublesome to get rid of them.

Good weekend to everybody.
- Emir

"Dima Klenchin" <klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu> wrote in
message news:961ti8$k78$1 at news.doit.wisc.edu...
> "Emir" <ekhatipo at NOSPAMmidway.uchicago.edu> wrote:
> :Hi,
> :I would appreciate someone recommending me a good strategy in finding
> :conditions to prevent destabilization and aggregation of the recombinant
> :proteins.
>
> The good strategy is to take protein in stable form (high enough or low
> enough salt, diluted enough, etc), and then shift it to conditions where
> protein without "protectant" precipitates. Then you can easily see
> effects of various additives.
>
> :I can express my 6-His tagged DNA-binding recombinant protein (RecA
> :homologue) in the range of 5 to 50 mg/l, depending on conditions and
E.coli
> :strains used (pET11 in BLR cells, induction with lambda CE6), as
estimated
> :by Westerns with cell lysates. I can extract most of the protein by
> :freeze-thawing, DNAse treatment, and sonication in 50mM HEPES, pH 7.5,
200mM
> :NaCl, 1mM DTE (+protease inhibitor cocktail + PMSF).
>
> First I would try increasing salt concentration to 0.5-1.0 M.
>
> :However, once in solution, the protein tends to precipitate, as seen by
> :increasing cloudiness of the extract, so that after IMAC on Talon resin
and
> :elution of the protein with a 100mM imidazole step I end up with only
~10%
> :of the protein, with the rest being not bound to resin, because of its
> :aggregation. It could be that DNAse treatment destabilizes the protein,
but
> :otherwise most of the protein will be pelleted along with the DNA during
the
> :clearing of the cell lysate.
>
> Again, rising salt might help here.
>
> :I tried adding 10% glycerol to the buffer, sucrose, PEG 3600, but it did
not
> :stabilize the protein.
> :Any ideas on what compounds to try to stabilize the protein? One of the
> :Creighton's lab manuals recommends PEG 1000, guanidine sulfate (not
> :chloride), betaines, TMAO, MPD (which is quite expensive), (NH4)2SO4,
MgSO4,
>
> Of these, my first choice would be betaine. Missing from the list
> are mild detergents. In your case, I would first try cholate.
>
> :If you had similar problems and succeeded in stabilizing your protein, I
>
> We had solubility problems with one E.coli expressed PH domain -
> it precipitated above certain concentration and precipitated whenever
> frozen in tubes. The only solution we found was to keep it in 20%
> ethylene glycol during purification, elute in large enough volume and
> freeze by letting 25 ul drops go directly into jar with liquid nitrogen
(great
> way to aliquote, by the way - you can scoop resulting beads, place them
> in cold eppendord and roll out one by one when necessary).
>
> Good luck!
>
>         - Dima







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