I am having issues restoring activity to an extradiol dioxygenase that I am studying. It is a recombinant form of a Rhizobium enzyme, expressed with a N-terminal 6xHistag in E. coli BL21 cells. I suspect that the Histag is somehow responsible for inactivity and I am in the process of cleaving it using thrombin.
I can't help but thinking that I am overlooking the fact that a cofactor is required by the enzyme. The same enzyme has been shown to work using the cell lysate and although my goal is to be using purified protein I can't even get the cell lysate to work. It is my understanding that LB media is supplemented enough to provide minute amounts of possible cofactors for growth. Additionally I supplemented the LB media with 1mMFeCl2 (ferrous iron binding site on enzyme) and still no activity. Previous work on this enzyme involved growing in YEM broth, which I can't imagine making a big difference.
Would using YEM media over LB make any difference in activity of my enzyme?
How does one usually determine which cofactor is required for an enzyme? Besides looking for conserved metal binding domains is it a good idea to create a cofactor "cocktail" and use that to determine activity?
Thank you for any words of advice.