I agree with the accolades given by Raji to the CCPBB, but suggest a difference: it is not given by "Nature," or any other polytheistic entity but rather by the patience and sustained efforts of all the souls who resist the urge to excoriate, which I would guess scales linearly to the number of years on the BB.
Thanks to all of you who help out for little to no reward, Jacob ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [email protected] ******************************************* ----- Original Message ----- From: Raji Edayathumangalam To: [email protected] Sent: Wednesday, February 25, 2009 2:01 PM Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli Did you not know that the CCP4BB-- even when all else appears to be melting down-- is Nature's sustained gift to humankind? Did you also not know that astronauts, astronomers, astrologers, artists, musicians, sportsmen, scientists, politicians, writers and everyone else subscribes to the board.... Given the breadth of knowledge of the CCP4BBers, molecular biology is still down the CCP4BBer's alley! Doesn't shock me that some molecular biologists responded :) The questions usually boils down to: To respond or not to respond? Cheers, Raji On Feb 25, 2009, at 2:48 PM, Mo Wong wrote: 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 <[email protected]> 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!
