How to keep recombinant protein stable and soluble?

[dustjohn at hotmail.com]

Can somebody please comment on the following:

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

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