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Re: [ccp4bb] Off topic: PDI overexpression in HEK293 cells
 

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CCP4bb <-- 2008 <-- April 2008 <-- 03 April 2008
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Subject: Off topic: PDI overexpression in HEK293 cells
From: Kenneth Verstraete kenneth {- dot -} verstraete {- at -} UGENT {- dot -} BE
Date: 2008-04-03 		Next message:
Subject: Re: twinned?
From: Bart Hazes bart {- dot -} hazes {- at -} UALBERTA {- dot -} CA
Date: 2008-04-03


Subject: Re: Off topic: PDI overexpression in HEK293 cells
From: VADIM A KLENCHIN klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2008-04-03
Kenneth,

> sorry for this off topic question. I am producing the ectodomain of a
>
> human receptor in HEK293T cells by transient expression. The
> expression
> levels are quite low. The protein contains a high number of disulfide
> bridges. It has been reported that overexpression of protein disulfide
> isomerase can enhance the secretion of proteins with a high disulfide
> content.

Since this is ectodomain, I presume it contains native sorting sequence.
In which case you don't have to worry about disulfide formation -
your protein will pass through golgi which is oxidative environment
favoring S-S bridges. (The PDI thing you mention probably refers to
E.coli; I've never heard about it in mammalian overexpressions).

With regard to low levels of expression: Is there a particular reason
to use 293 cells? Transient expression in Cos-1/Cos-7 cells gives
~ 10 fold higher levels of expression because of the plasmid amplification
in these cells. (The only requirement is that the plasmid should
have SV40 ori; most mammalian vectors do).

Dima

CCP4bb navigation

CCP4bb <-- 2008 <-- April 2008 <-- 03 April 2008
Previous message:
Subject: Off topic: PDI overexpression in HEK293 cells
From: Kenneth Verstraete kenneth {- dot -} verstraete {- at -} UGENT {- dot -} BE
Date: 2008-04-03 		Next message:
Subject: Re: twinned?
From: Bart Hazes bart {- dot -} hazes {- at -} UALBERTA {- dot -} CA
Date: 2008-04-03


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