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

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From: Pius Padayatti ppadayatti {- at -} GMAIL {- dot -} COM
Date: 2008-03-05

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Subject: Re: Removal of glycosylation sites in Picha expression construct
From: hua jing jing_hua {- at -} HOTMAIL {- dot -} COM
Date: 2008-03-05

I don't know much about Pichia expression system. You can consult glycobiologists if you are 100% sure that no mistake was made during the experiments.

Our lab has been using Drosophila S2 insect cell expression system. With this system, you can maintain cell culture using roller bottles or flasks (so better than the baculovirus expression system). The N-linked glycan is about 1000 dalton per site which can be removed easily by enzymes. The yield is typically above several mg per liter for even large proteins (>80 kDa).

Holly
----------------------------------------
> Date: Wed, 5 Mar 2008 08:38:46 -0600
> From: Radisky.Evette@MAYO.EDU
> Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
> To: CCP4BB@JISCMAIL.AC.UK
>
>
> Thanks to everyone for the great suggestions so far. To clarify and
> answer a few questions, with the mutated construct we get no protein
> either secreted or in the pellet. The protein in question is about 20
> Kda; with glycosylation at both sites it is around 29 kDa (both in
> mammalian cells and in Pichia). The protein has been previously
> produced and crystallized by another lab; in that case the double Ala
> mutant was expressed in CHO cells. It has been shown that glycosylation
> is not required for function. There are several reports of related (but
> natively nonglycosylated) proteins being produced in Pichia. We decided
> to try Pichia because we were already using it for another project, and
> we decided to introduce the Ala mutations because that mutant protein
> had apparently been crystallographically well behaved previously.
>
> Thanks again,
> 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: Off topic: General rule for maximum flow rate for affinity column?
From: Pius Padayatti ppadayatti {- at -} GMAIL {- dot -} COM
Date: 2008-03-05

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Subject: Neutron Protein Crystallography Call for Proposals New Deadline
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