Evette,
As the glycosylation seems to be critical for production of your
protein in P. pastoris I would imagine it would be the same in both
insect and mammalian cells. As I'm sure you've invested plenty of time
getting this expression system to work it would seem a waste not to try
enzymatic treatment with either Endo H or PNGaseF (although you may
need to denature the protein to get complete removal with the latter
enzyme)
I'm curious about your choice of mutations though. Although I know
it's standard to perform Alanine substitution, switching from a known
surface Asn to an Ala seems pretty drastic to me. If I perform a
"google" survey of the literature it seems mutating the Asn to an Asp is
an alternative (and better ?) choice, I also have seen publications
where mutation of the Ser/Thr residue of the N-glycosylation
recognition site to an Alanine appears to work. I also agree with
others on the CCP4 bb of trying the mutations individually.
Stephen
--
Stephen Weeks, Ph. D.
Drexel University College of Medicine
Department of Biochemistry and Molecular Biology
Room 10102 New College Building
245 N. 15th St.
Philadelphia, PA 19102
Phone: (+) 215-762-7316
Fax: (+) 215-762-4452
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)
