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

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From: Mike Latchem mike {- dot -} latchem {- at -} DIAMOND {- dot -} AC {- dot -} UK
Date: 2008-03-05

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Subject: Re: Removal of glycosylation sites in Picha expression construct
From: "Radisky, Evette S {- dot -} , Ph {- dot -} D {- dot -} " Radisky {- dot -} Evette {- at -} MAYO {- dot -} EDU
Date: 2008-03-05

Subject: Re: Removal of glycosylation sites in Picha expression construct
From: Stephen Weeks stephen {- dot -} weeks {- at -} VERIZON {- dot -} NET
Date: 2008-03-05

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)
>

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

Previous message:
Subject: Re: Copper Staining
From: Mike Latchem mike {- dot -} latchem {- at -} DIAMOND {- dot -} AC {- dot -} UK
Date: 2008-03-05