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CCP4BB  February 2009

CCP4BB February 2009

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Subject:

Re: Off topic: Mammalian gene expression in E. coli

From:

Stephen Weeks <[log in to unmask]>

Reply-To:

Stephen Weeks <[log in to unmask]>

Date:

Tue, 24 Feb 2009 19:47:59 -0500

Content-Type:

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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. Iíve 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). Iíve 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 
> Iím not sure they examine putative mRNA substructure formation like 
> some companies do). Itís only 49c per base pair, so doesnít 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!

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