endotoxin in recombinant proteins?

Ross Prestidge R.Prestidge at genesis.co.nz
Mon Jul 7 17:50:25 EST 1997


Can anyone point me to a FAQ on the problem of endotoxin contamination
in proteins purified from recombinant E. coli?   I am working with a
gene which I have expressed from E. coli as a fusion with a His-tag.  
The tagged protein has been purified on a nickel column, but at this
stage has slightly too much endotoxin for my application.   My questions
are: 

- Has anyone investigated the best washing conditions for nickel columns
to minimise endotoxin carryover?

- What is the best source for endotoxin analysis kits?   At present I am
using the COATEST LAL kit from Chromogenix ( manufactured by Endosafe),
and it's working well, but I would be interested in a cheaper
alternative.

- What is the best way to remove endotoxin from purified recombinant
proteins?   I have some Pierce Detoxigel, but I haven't tried it yet.  
I would be interested to hear of your experience with this matrix,
optimal buffers for protein work, alternative resins etc.   My protein
is quite hydrophobic - is there a risk it will bind to the resin?

I would be grateful for any help you can give, either by mail or to the
newsgroup.
Regards,
Ross Prestidge



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