We haven't tried SUMO, but had some frustrating results with GST fusions. They did improve expression and solubility - BUT in one case the target protein precipitated immediately when the tag was cleaved off, and resisted all attempts to bring it back to life. In another case, the fusion protein dragged chaperones into the prep that were nearly impossible to get rid of completely, thus ruining our ATPase assays.
Is SUMO, being smaller, less likely to drag such crud along with it? Phoebe ---- Original message ---- >Date: Wed, 25 Feb 2009 14:48:57 -0500 >From: Mo Wong <mowon...@gmail.com> >Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli >To: CCP4BB@JISCMAIL.AC.UK > > 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! Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
