Nothing out of the ordinary there. You can try to induce overnight (for 20 hours or so) at 15C. And you can try to reduce the concentration of IPTG if excess protein is going to inclusion bodies anyways. Collect whatever protein is soluble and ignore your inclusion bodies. The His tags allow you to capture the protein easily from large batches of cell culture and cell lysate. As long as you get some soluble protein in good condition, you could just make more of it and not worry too much with optimizing the expression conditions.
Jose. "Buz Barstow" <b...@mac.com> 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 > -- *************************** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***************************
