pGEX-fusions

Gordon Munro munro at sbsu1.aukuni.ac.nz
Sun Jul 4 00:31:33 EST 1993


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

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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