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

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From: Thomas Eriksson thomas {- dot -} eriksson {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
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Subject: Re: Removal of glycosylation sites in Picha expression construct
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-03-05
If you have poor conservation in this region and the region itself is a
loop, I would perhaps attempt to replace the entire loop with another one -
from a homologue that does not have the glycosylation site(s). Not that this
is guarranteed to work, but it might.

Artem

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Radisky, Evette S., Ph.D.
Sent: Wednesday, March 05, 2008 9:39 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression
construct


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)

CCP4bb navigation

CCP4bb <-- 2008 <-- March 2008 <-- 05 March 2008
Previous message:
Subject: Re: Missing fonts...
From: Thomas Eriksson thomas {- dot -} eriksson {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2008-03-05 		Next message:
Subject: Re: Diamond and ESRF future.
From: John R Helliwell jrhelliwell {- at -} GMAIL {- dot -} COM
Date: 2008-03-06


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