[ccp4bb] Removal of glycosylation sites in Picha expression construct

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: multiple sequence alignment from multiple pairwise structural alignments
From: P Hubbard dienoylcoa {- at -} HOTMAIL {- dot -} COM
Date: 2008-03-04

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Subject: Re: Removal of glycosylation sites in Picha expression construct
From: artem {- at -} XTALS {- dot -} ORG artem {- at -} XTALS {- dot -} ORG
Date: 2008-03-04

Subject: 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: 2008-03-04

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)

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: multiple sequence alignment from multiple pairwise structural alignments
From: P Hubbard dienoylcoa {- at -} HOTMAIL {- dot -} COM
Date: 2008-03-04

Next message:
Subject: Re: Removal of glycosylation sites in Picha expression construct
From: artem {- at -} XTALS {- dot -} ORG artem {- at -} XTALS {- dot -} ORG
Date: 2008-03-04