Thanks to all who responded. Actually, this bulletin board is better for help with molecular biology than the molecular biology bulletin board I am subscribed to!
On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks <stephen.we...@verizon.net>wrote: > Mo, > Just to add my 50 cents, I didn't see any mention of the use of fusion > proteins in your original post. GST, MBP or my personal, and completely > biased, favourite SUMO (plus many more proteins) have been shown to enhance > expression when fused to the amino terminus of a target protein. If you fear > you have toxicity, simply tracking the OD600 pre and post induction normally > tell you if this is happening. I've worked with proteins that basically > baselined the cell growth upon induction and, as Artem stated, at least I > knew my protein was being made albeit at very low levels. > > Stephen > > -- > Stephen Weeks, Ph. D. > Drexel University College of Medicine > Department of Biochemistry and Molecular Biology > Room 10102 New College Building > 245 N. 15th St. > Philadelphia, PA 19102 > > Phone: (+) 215-762-7316 > Fax: (+) 215-762-4452 > > > > Mo Wong wrote: > >> 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! >> >
