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Protein toxity in E.coli

Zhonglin Chai zchai at COBRA.PATH.MONASH.EDU.AU
Sun Jun 29 22:14:32 EST 1997


aplummer at brain.UCCS.edu (AL R. PLUMMER) wrote:
>Our lab is trying to overexpress and isolate a rather large yeast (S.
>cerevisiae) protein in E.coli.  The gene is ca. 4 kb and encodes a protein
>of 1,433 a.a.  We transformed DH5alpha with a plasmid containing the
>gene, but are unable to see it.  Also, when the expression is induced
>(IPTG), the E.coli become sick (grow slower).  Does anyone have any
>experience expressing large or toxic proteins in E.coli?  Any help
>would be greatly appreciated.
>
>-Al (plummer at em.uni-frankfurt.de)

I posted the similiar questions recently, here are the replies I received
(I did express some proteins, but mostly degraded, in BL21(DE23)pLysS cells
(Promega) at room temperature overnight):

Date: Fri, 30 May 1997 19:14:17 -0700
From: Michael Kalchman <kalch at ulam.generes.ca>
Reply-To: kalch at ulam.generes.ca
Organization: UBC Medical Genetics
MIME-Version: 1.0
Newsgroups: bionet.molbio.methds-reagnts
To: Zhonglin Chai <zchai at cobra.path.monash.edu.au>
Subject: Re: Fusion protein toxic to E. coli.?

A couple of things:

1.  Check your clone for repeats
2.  Ensure you are using a good strain of bugs, e.g. AD202, BL21, or
UTC5600

Good luck

Michael

*****
Sender: willis at gandalf.psf.sickkids.on.ca
Date: Fri, 30 May 1997 11:11:06 -0400
From: Randy Willis <willis at gandalf.psf.sickkids.on.ca>
MIME-Version: 1.0
Newsgroups: bionet.molbio.methds-reagnts
To: Zhonglin Chai <zchai at COBRA.PATH.MONASH.EDU.AU>
Subject: Re: Fusion protein toxic to E. coli.?

Zhonglin Chai wrote:
> I did observe that the E.coli. cell density dropped soon after IPTG
> induction.

Zhonglin,

Toxicity of a foreign protein does occur on a regular basis in E.coli
although I must admit that this is the first time I've heard it for GST
fusions.  In the past, whenever I have come across this problem, I have
been forced to do the inductions at much lower temperatures.  I am
working on the assumption that this means that the protein is expressed
at a much lower rate which allows the cells time to deal with the alien
intruder.  The following protocol may be of assistance although you may
have to try variations of this for your system:

inoculate and grow at 37C
at OD600=0.4, change temp to 30C
at OD600=0.6, change temp to 25C
at OD600=0.8, change temp to 20C
at OD600=1.0, change temp to 15C
at OD600=1.2, induce with IPTG
grow for 8-36h at 15C, taking time points for induction and OD

(unless you work in a really cold room, I would suggest moving the
shaker into the cold room for growths at 15C)

Another possibility is to change your fusion system to something which
has a tag which promotes precipitation of your protein in the cell into
inclusion bodies.  This may assist the cell in dealing with your
protein.  The only problem, of course, is then being certain of
refolding at least enough to cleave the tag.  In the pET system from
Novagen, there is a T7 tag which can be added and this is from the T7
coat protein and promotes pptn.

Good luck and let us know how things go.
Randall C Willis, Researcher
Biochemistry Research, Hospital for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8  CANADA

416-813-5933 (ph)  -5022 (fax)
willis at gandalf.psf.sickkids.on.ca

******
Date: Sat, 31 May 1997 13:20:20 -0700 (PDT)
From: Doug Pecota <pecota at eng.uci.edu>
To: Zhonglin Chai <zchai at cobra.path.monash.edu.au>
Subject: Re: Fusion protein toxic to E. coli.?
MIME-Version: 1.0

Six suggestions:

1. Induce for less time.

2. Add additional antibiotic when you induce.

3. Check plasmid stability before induction. If not 100%  perhaps use a
   tighter controlled promoter or catabolic repression during earlier stages of
   growth (eg add glucose which you dilute a way before induction.)

4. Isolate a strain that is resistant to your toxic protein but does not
   degrade it.

5. Add a post-segregational killer loci to you plasmid so that plasmid
   free cells will not take over the culture so rapidly.

6. Try growing in a protease deficient host.

Hope this helps.

--------------------------------------------------------------------
Doug Pecota
University of California Irvine
pecota at eng.uci.edu

It isn't just what you know,
nor even who you know,
but also what you know
that isn't so.
--------------------------------------------------------------------

******
Date: Sat, 31 May 1997 21:43:58 -0400
From: barrs01 at doc.mssm.edu (Sharon Barr)
To: zchai at COBRA.PATH.MONASH.EDU.AU (Zhonglin Chai)
Subject: Re: Fusion protein toxic to E. coli.?
Organization: Mount Sinai School of Medicine

We had a similar problem, although to a lesser degree.  Our problems were
solved by changing bacteria strains.  We now use BL21-LysS, which is
tolerant of toxic proteins and lyses easily with one freeze-thaw.  Why not
give it a try?  You might also check out a recent biotechniques paper,
although I cannot remember the authors or issue #(May 1997).  They
described a very similar problem, and werew able to recover higher levels
of their toxic protein if they did NOT induce with IPTG.  Just something
to think about.

-Sharon Barr
Mount Sinai School of Medicine
Box1194
NY, NY 10029
barrs01 at doc.mssm.edu

In article <v01530501afb439c17795@[130.194.152.101]>,
zchai at COBRA.PATH.MONASH.EDU.AU (Zhonglin Chai) wrote:

> Hello netters,
>         I have been experiencing difficulties with expression of a GST
> fusion protein with a human cDNA (120kDa). I am using a pGEX vector that
> was very good for expression of the C-terminus of the same protein. I
> simply could not obtain any fusion proteins detected by either SDS-PAGE, or
> glutathione beads purification (no protein eluted from the column).
>         I did observe that the E.coli. cell density dropped soon after IPTG
> induction. I also try to induce with a low densith of cells with a parellel
> control without IPTG induction. After 4-5h, The flask without IPTG gave a
> much higher (probably 20 times higher) E.coli. density than that induced
> with IPTG.  I thought that most of the bacterial cells had been dead and
> probably lysed by the expressed fusion protein (not good), otherwise the
> cells were growing extremely slow because they were making a lot of extra
> fusion proteins (good thing) for me). However I  left the IPTG induced
> bacteria being shaken at 37degree C. O/N. and they reached the saturate
> density in the following morning. I tried SDS-PAGE (Commassie blue
> staining) and glutathione beads purification with the cells. The same
> result, no fusion protein.
>
>         I would appreciate any suggestions and commends.



Dr. ZhongLin Chai
____________________________________________
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9276 2698 (lab)
           (61 3) 9276 2696 (office)
           (61 3) 9384 1305 (home)
           0419 87 1940 (mobile)
           + 61 419 87 1940 (international mobile)
Fax:       (61 3) 9529 6484
email:     zchai at cobra.path.monash.edu.au
____________________________________________





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