Hello All,

I am expressing a recombination protein containing a His10-GFP tag connected to my protein of interest via a 3c cleavage site. The idea is to pull down with talon resin, elute with Imidazole, dialyse away the imidazole, cleave, rebind the free His10-GFP using talon, then finally elute pure protein of interest. The problem is that after the initial binding step and washing, I'm eluting my protein of interest (with imidazole mind), but as the free pure protein. I'm somewhat baffled, but the only conclusion I can come up with is that non-specific proteases must cleave on bead. I elute with an imidazole buffer without protease inhibitors. But, everything before this has protease inhibitors (Sigma's protease inhibitor cocktail). I've never worked with yeast before, but could there be proteases in X-33 and GS115 strains that could cleave a 3C site, or elsewhere on GFP.

Thanks in advance and I look forward to your responses.

Tony