Print

Print




Concerning the contamination of recombinantly expressed proteins with bacterial chaperones, one might wish to try a complete removal using ATP and denatured protein as a "bait" as described here:
 
Protein Expr Purif. 2000 Oct;20(1):45-7.
Separation of copurifying GroEL from glutathione-S-transferase fusion proteins.
Rohman M, Harrison-Lavoie KJ.

Might work or not but could save a lot of time consuming search for the right fusion-tag.

Florian



Phoebe Rice wrote:
[log in to unmask]" type="cite">
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 <[log in to unmask]>  
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
    
E. coli  
  
To: [log in to unmask]

  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
  <[log in to unmask]> 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