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)
