Recently, i've been trying to purify my protein of interest. The protein size is approximately 160KDa and i can purify this in mammalian cells by using Flag tag system. Now, I wanted to get enough proteins.. so I used bac to bac system. However, I can see my protein of interest only in insoluble fraction.. I've tried many detergents such as 1% Triton X-100, 1% OG and 1% cholic acid but still I cannot solublize this protein.
The thing I cannot understand is that I can solublize this protein in mammalian cells overexpressing this but i couldn't get this in sf9 cells.
This protein is an enzyme so I'm afraid to use Gn-HCl.. it'll be okay to purify an enzyme?
Is there anybody knows about this? and any solution?
If anyone gives solutions and answers about this, it'd be great help to me.
insoluble protein in sf9(but soluble in mammalian cells)