Dear All:
I have a peculiar problem with baculovirus expression of my protein. The
native protein elutes in Tris gradient. This has no problems.
However, a mutant elutes in the wash. After further purification with
size exclusion, I notice that this mutant is always associated with
some medium component that I cannot separate. I know this (?) because
when lyophllized (not a sin for this protein), I get 15-30 mg
of protein (or colorless fluffy material) whereas protein estimation
gives only about 5 mg. This amount is what I usually get for the native
protein.
I have tried active charcoal, BioRex, ammonium sulfate precipitation and
even hydroxyapatite columns but nothing seems to work to
separate the extra material from the protein.
Any insight would be of much help.
Thanks a lot
Subbu
