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
