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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!