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

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From: Kay Diederichs kay {- dot -} diederichs {- at -} UNI-KONSTANZ {- dot -} DE
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Subject: Re: Removal of glycosylation sites in Picha expression construct
From: Rob Meijers ccp4spamalot {- at -} YAHOO {- dot -} COM
Date: 2008-03-05

Subject: Re: Removal of glycosylation sites in Picha expression construct
From: Vangelis Christodoulou e {- dot -} christodoulou {- at -} NKI {- dot -} NL
Date: 2008-03-05

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)
>

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CCP4bb <-- 2008 <-- March 2008 <-- 05 March 2008

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