This is not entirely uncommon. Did you try removing just one of the two sites (sometimes it helps) - combined with enzymatic deglycosylation this may give you good enough protein to work with.
If you try other hosts, I would definitely consider Schisosaccharomyces pombe - its glycosylation patterns are much simpler. Other likely candidates include regular bakers' yeast and Hansenula polymorpha. I would try both the native and the mutant proteins in those hosts. There are tricks one can play with Pichia to try and make it express the deglycosylated protein but it almost sounds like you've hit on something very essential since you're going from good expression down to no expression. Lastly, you could try insect cells :) Artem > > Dear all, > > Our lab is new to working with Pichia pastoris, also new to working with > glycosylated proteins. We have a construct for a secreted protein that > expresses pretty well in Picha, but upon mutation of the 2 N-linked > glycosylation sites to Ala, we get no expression at all, nada. The > nucleic acid sequence appears to be correct, i.e. we have not introduced > any unintentional frame shifts, stop codons, or anything like that. Is > this a common phenomenon? Are there any tricks to get the Pichia to do > its thing? Any chance that alternative substitutions will work when Ala > does not? Or are we better off (a) trying to deglycosylate > enzymatically, or (b) trying a different expression host? All opinions > and anecdotes welcome. > > Thanks! > Evette > > Evette S. Radisky, Ph.D. > Assistant Professor and Associate Consultant II > Mayo Clinic Cancer Center > Griffin Cancer Research Building, Rm 310 > 4500 San Pablo Road > Jacksonville, FL 32224 > (904) 953-6372 (office) > (904) 953-0046 (lab) > >
