Hi Petros, It's possible but unlikely that your media contains enough metal ions to matter. More likely options include protein issues like overgkycosylation, proteolysis (removal of the tag) or severe aggregation. Since you are not experiencing this in BMGY, I wonder why not to supplement your synthetic medium with yeast extract and/or peptone and the like?
Artem On Mon, Mar 26, 2012 at 7:04 AM, Petros Giastas <[email protected]> wrote: > Dear all, > > I am expressing a 6xHis tagged secreted protein in a fermentor in P. > pastoris, using the standard minimal medium described in the invitrogen > manual (plus PTM1). Following collection of the culture medium, I am having > problems with purification of the protein as only a small fraction (~10%) > binds to the Ni-NTA beads even after extensive buffer exchange (when > expressed in full BMGY media this is not observed). Could this be attributed > to metal ions still present in my sample? Is it likely to be due to poor > protein quality in this medium? Or any other suggestions? > > Thanks in advance > Petros
