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Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
 

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CCP4bb <-- 2008 <-- March 2008 <-- 05 March 2008
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Subject: Re: Removal of glycosylation sites in Picha expression construct
From: Vangelis Christodoulou e {- dot -} christodoulou {- at -} NKI {- dot -} NL
Date: 2008-03-05 		Next message:
Subject: Re: Problem with DM
From: "P {- dot -} J {- dot -} Briggs" p {- dot -} j {- dot -} briggs {- at -} DL {- dot -} AC {- dot -} UK
Date: 2008-03-05


Subject: Re: Removal of glycosylation sites in Picha expression construct
From: Rob Meijers ccp4spamalot {- at -} YAHOO {- dot -} COM
Date: 2008-03-05
Dear Evette,

it is quite common that mutation of glycosylation sites completely kills
expression. One rationale is that the glycans cover hydrophobic patches
on the surface of the protein, and when exposed, the protein won't fold
properly anymore.
Pichia is a pain because the glycans it produces are enormous and
heterogenous. That means that when you deglycosylate, you end up
with a protein sample that has glycans of varying length which certainly
does not help in crystallization.
There are some commercial pichia strains out there with a modified glycosylation machinery. But I would certainly commend Artem's suggestion to switch to baculo.

Cheers,

Rob Meijers
Synchrotron Soleil

"Radisky, Evette S., Ph.D." wrote: Removal of glycosylation sites in Picha expression construct
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)



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