Mammalian protein expression in E. coli - (Feb/24/2009 )

A problem as old as molecular biology itself....

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 should probably 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, for folks who have had similar problems, would you recommend that I first try this over testing protein expression in yeast/insect cells, or the other way round?

Thanks!

If the protein is toxic, you would not see much growth, which you can check by taking ODs after induction.
General questions: What vector are you using? How bad is the codon bias in terms of strings of low-frequency codons? Are there strings of low-freq codons?

For gene synthesis, we have used Geneart and that has worked OK.

If you've tried bacteria a few times, I'd consider moving organism, but you're going to have to re-optimise again (I guess you're well aware of that though...)