How to keep recombinant protein stable and soluble?

Artem Evdokimov AEVDOKIMOZ at
Tue Nov 12 18:28:41 EST 2002

> After capturing a his-tagged protein on Ni-NTA, it is eluted with 250mM
imidazole, 500mM NaCl and 50mM sodium phosphate at pH=8.0.  The protein in
this sample appears to be soluble (although I've never left it for extended
periods of time) but after 3 rounds of concentrating via centrifugation in a
centricon device and buffer exchange (to a low-salt buffer for anion
exchange), there are large aggregates of protein that have formed.  After
addition of 20mM 2-mercaptoethanol and incubation on ice for 10 minutes,
much of the aggregated protein disappears.  Then subsequent anion exchange
yields his-tagged protein that elutes at 250mM NaCl; this sample once again
aggregates if left at 4 celsius overnight.  Are there any secrets to keeping
such a protein soluble?  Should we replenish reducing reagent continually?
Should we add glyercol or switch to a slower dialysis period?
> Thanks,
> Dustin

1) Perhaps your protein likes to be in high salt ? Need to check.
2) Use DTT in all buffers and freeze the protein ASAP
3) do you have activity check to see if the protein that you brought back
from the dead using reducing agent was actually alive ?

More details would help - what's the pI of the protein, how many cysteines,
is it pro- or eukaryotic, etc.


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