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
