pGEX-fusions

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Mon Jul 5 14:56:00 EST 1993


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sbhattac at crc.ac.uk (Dr. S. Bhattacharya) writes:

>Hi
>I'm affinity purifying a GST-fusion protein expressed in E.coli using a 
>Glutathione-sepharose 4B matrix from Pharmacia.  I get a nice 52 kd band
>which I've blotted and is a fusion protein as it is detected by an anti-GST
>antibody.  I also get a 69 kd band co-purifying; this is not a fusion
protein.
>It does not appear when I express the GST alone. Changing the cell type
>(DH5alpha and TOPP-1) does not affectit.  Washing the matrix (before elution)
>with increasing stringency of NaCl (upto 1M) also does not help. Pharmacia
>tell me that this hasn't been reported to them before. 

>Has anyone else had a similar experience, I would be glad to hear.  Also any 
>suggestions on how I can get rid of the extra band would be much appreciated.

>Shoumo Bhattacharya
>MRC Molecular Medicine Group
>RPMS London

>I was interested to read of your proble and am sorry that I have no
>suggestions
>,  but I have a proble of my own with pGEX-KG which I wonder if anyone can
>help.

>My fusion protein is 70kDa,  but if I run a sample of th glutathione purified
>fusion on a SDS PAGE gel,  I get 2 bands,  one at 70 kDa as expected and one
>at 26kDa (about the right size for GST).   Unfortunatley the smaller band
>is by far the more prominent one sugesting that most of the bacteria in the 
>culture are only producing GST, not the fusion.
>I start with a single colony known to contain the fusion and have not treated
>the fusion with thrombin prior to sds-page,  so theoretically there should be
>no GST produced at all.  Has anyone had any expersience with  spontaneous
>loss of th
>their insert in pGEX-KG?  Or does anyone know if the fusion protein is
>unstable?

>Any comments/ideas appreciated

>Gordon Munro
>School of Biological Sciences
>Auckland University
>New Zealand
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Hi Gordon!
    Are you sure that this is the case, spontaneous loss of your insert
that is.  It would seem more likely that what you have is a cell or a culture
with two plasmids segregating.  Streak for singles, pick 4 or 5 or 6 colonies 
and 
do a mini experiment to see if this is infact the case. You might even want
to grow non-selectively for a while make sure that the plasmids segregate.
Another likely possibility is perhaps your plasmid is a dimer.  Direct repeats
are viable in coli.  One of the repeats has no insert and the other has your
gene fusion.  This type of plasmid might go undetected depending on the cut 
you used to find it from a miniprep gel.  I would also
check to see if your reading frame is right!  This may be way off base but 
most
of the strains we work with are SupE or SupF or some other suppressor tRNA. 
Could it be that you have a stop codon inbetween your genes which is being
suppressed at a high enough frequency to see protein on a gel? Does anyone
have any data on this type of thing? 
Another more serious interpretation is that this fusion is the dnaK gene 
product.
and not your fusion protien at all.  Recently some one posted a message
saying that glutathione agarose (sepharose) also binds the dnaK gene 
product from coli,
I remember it being about 70 kd. ( Is this right?)  I trashed my copies of 
that
message so I can't even check this.   This may be what Shoumo
 Bhattacharya is seeing too (69kd).  Shoumo mentions that this 
band is not evident in the pGEX only strain, probably meaning that GST is 
produced
at levels much higher than the fusions competing out all else for a spot on 
the
beads.  In all our studies with pGEX-2T I don't remember seeing
this band but we use Sigma glutathione agarose not pharmacia 
Glutathione-sepharose 4B matrix. I'm not sure if this makes a difference.
We also use JM109, which seems to be better that DH5alpha.  I wonder if this 
70kdb
band is present in a bacteria only negative control?  

Just some thoughts! 

Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________




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