Help!recombinant Protein degradation during purification - Proteolysis (Sep/09/2005 )

Hi ,

I'm a brit so what be on at the same time as you so will reply at weird times. I'm new to this protein purification game and am hitting some problems. I have a protein encoded in pET 15b plasmid produced by Rosetta 2 expression system. The expression is good and binding to my Talon column (via His tag) and then binding to an anion exchange column is good. The problem is that I lose a lot of protein during lysis procedure (and even during expression in vivo) and also while loading onto Talon column. After recomb protein has been initially purified using talon column degradation stops but by then I have already lost far too much protein.

To my lysis buffer I add
*B-mercaptoethanol 1mM
*PMSF 1mM
*EDTA free protease inhibitor tablets (not sure what they contain as company is quite protective over recipe, but they do help)
any ideas as to what else I could add, I don't want to denature protein and I need it active.

Thanks for any replies
Ali

p.s. anyone know of any agents I could add to expression culture to prevent foaming of culture which is reducing my aeration. Thanks

Have you checked if pH of your buffer is far away from the pI of your protein

If not, protein will precipitate and you will loose it

So far, now I have also problem with expression of my protein in Rosetta2. Have you just Rosetta 2, or maybe Rosetta 2 DE3 pLacI, which is, i am not sure, but maybe is more convenient for the pET vectors?