I am trying to express and purify a membrane protein in HEK293T cells that has a tag containing His and Rho1D4. The protein does express (binding activity and positive signal on Western blot are there; not for the tags though). But I get no binding to any of the columns at all. All protein is in the Flow-through. I also have a factor Xa cleavge site there. So is it possible the tags get cleaved off by the HEK293T cells? Also, I am using FBS, does that contain active Factor Xa?
Kerstin, you presumably don´t need the factor Xa cleavage site except you want to use the protein for crystallization studies or for any studies where the tags might interfere. Your data seem clear and answer your question. Obviously there is an active factor Xa in your system...
Commercial Factor Xa is actually purified from bovine serum but normally requires activation. I assume you could try washing your cells in DMEM or PBS/EDTA before extraction but if your protein is a membrane protein and the Xa site is already exposed on the extracellular face of the cell membrane it may be cleaved before you get that far. Do you know the expected topology?
Both His and Rho tags are removed by Factor Xa and if they are c-term are they definitely in frame with your gene?
If you still want to retain the tags see if anyone has serum-free mammalian cells (almost certainly in suspension) in your institute.
There are also inhibitors available specific to Xa but I suspect that they woudl be way too expensive to add to cells.
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