Buz, How big is the protein? How many metal binding sites are there? Do you know what metal it should bind? What is your media?
I am assuming it is a zinc binding protein, some zinc binding protein will not fold without enough zinc in the media. Perhaps you should not rule out refolding. ray USF On May 4, 2010, at 5:11 PM, Buz Barstow wrote: > Dear all, > > I am trying to purify a metalloprotein (a hydrogenase) using affinity > chromatography. > > I have produced two tagged versions of the enzyme: one with an N-terminal 6x > histidine affinity tag, and the other with a C-terminal 6x his-tag. The > tagged proteins are both tied to an IPTG-inducible promoter. > > When trying to express and purify the N-terminal tagged protein, I have found > that almost all of the expressed protein goes into inclusion bodies when the > culture is grown at 37 or at 30 degrees C. When the culture is grown at 20 > degrees C, a small amount of protein can be found in the cell extract. > > Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we > do not believe that the protein can be refolded from the inclusion bodies. > > Could you offer some advice on how to express and purify this protein and > reduce the quantity of protein found in inclusion bodies? > > Thanks! and all the best, > > --Buz
