Hi Evette,
Have you tried to mutate one glycosylation site at the time and check
for expression? Often glycosylation is necessary for secretion -that's
the case with my protein. Also these sites tend to have a defined
glycosylation pattern which eliminates the need of deglycosylation.
You could try a mammalian expression system as an alternative. I have
been using Invitrogen's Flp-In system to generate stable cell lines in
HEK293 Flp-In cells and has worked very well in my hands (I'm not
affiliated with Invitrogen).
Hope this helps.
Vangelis
On Mar 4, 2008, at 22:25, Radisky, Evette S., Ph.D. wrote:
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)
