I'm expressing a protein in S. cerevisiae, and I see it when I do a crude total extract. However, when I refine the protein extraction method, in order to obtain soluble proteins, I do not find my protein (Coomassie staining). I think it may be insoluble, since it is large - about 1200aa - and I'd like to talk with somebody who has encountered insoluble recombinant proteins when expressing them in S. cerevisiae.
I had just one case where I expressed a small human protein (expressed as inclusion bodies in coli)
in Pichia. In that case we observed that the protein was expressed as insoluble aggregates and left that system.
In your case, just to rule out other possibilities (i.e. rapid degradation of your protein during preparatio of the crude extracts), I would suggest to check for its presence in the insoluble fraction of the cell lysate by SDS-PAGE/WB.
Gianluca
Is your protein of interest possibly a membrane protein or membrane associated protein?
Thank you both for the comments.
Gianluca: I've already dissolved the pellet with urea, but I haven't checked for the presence of the protein there...
William: I have no indications that it will associate to the membrane when expressed in yeast, but I also can't exclude it... But it is supposed to be cytoplasmatic.
Are you getting complete cell lysis? I would carry out a western blot on whole cell lysis as well as supernatant after fractionation. This may tell you where your protein has gone. Note you will need extra SDS in the SDS-PAGE buffer to extract all proteins from the whole cell lysis fraction.
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