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aggregation of purified proteins - (Jan/16/2005 )

I am expressing recombinant, His-tagged proteins in Pichia pastoris and, after purification via Ni-NTA resin, the proteins aggregate very quickly upon storage at 4 degrees.

Can anybody out there suggest any generic methods to prevent aggregation of purified recombinant proteins.


Storing under denaturing conditions helps- but only if your protein will be used for antibodies. If this is an enzyme - try flash freezing the sample. Aggregation at 4C suggests psychrophobic properties.


Such problems occure with his-tag appended proteins. try storing the protein with 5% glycerol. or remove his tag


Depending on the original source of the protein, if it is recombinant, it may need to be stored at room temp. ALso depending on the elution process used pH or imidazole or even EDTA can all precipitate the protein. Exchange the buffer ASAP and dilute the protein. If you have very high protein concentration the protein can precipitate, that is how crystals are grown. If the protein was secreted, maybe add reducing agent.
I work on archeal and bacterial proteins mostly and they all like NaCl around 500mM and DTT. ALso stay at least 1pH unit away from pI and add possible cofactor or substrate, which all help the protein stay folded.


Try a KCl 150mM, KH2PO4 50mM buffer. I posted a message about this in reply to someone earlier. You might wanna check it out. Good luck.