I thought I'd post this to the CCP4bb, as judging by previous posts, it seems I could get some useful insight into my problem...
This
is question has probably been asked by people for a long as molecular
biology has been around, but hopefully my question isn't a complete
rehash of other peoples: I am trying to express a human protein in
bacteria where the only modified amino acids are 3 phosphorylated
serines. I’ve gone through the usual hoopla of trying to get it
expressed in E. coli (Rosetta/Codon+ cells, varying IPTG, low
temperature, etc). Sequencing confirms my insert is correct, but from
coomassie gel inspection, I appear to get near zero induction (I need
to do a Western to get a clearer assessment). I’ve heard about custom
gene synthesis, and it appears Mr. Gene (https://www.mrgene.com/) would
be a good avenue to look into as they optimize the ORF taking into
account codon usage in E. coli (though I’m not sure they examine
putative mRNA substructure formation like some companies do). It’s only
49c per base pair, so doesn’t seem too cost prohibitive. My only
concern is that if this protein is toxic, I could be wasting money.
So I was wondering, has anyone seen the expression for a particular protein change from zero in Rosetta/Codon+ cells using "native"
sequeneces to being largely overexpressed in BL21(DE3) cells using codon optimized sequences?
For folks who have had a similar problem to the one I've described,
would you recommend that I first try using a codon optimized sequence in
E. coli over testing protein expression in yeast/insect cells, or the
other way round?
Thanks!