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
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