bacterial expression/protein purification

Steven L Goldberg steven.goldberg at bms.com
Tue Apr 18 10:23:07 EST 2000


We have had good luck growing cells at lower temperatures than 37oC (as low
as 15oC).  The slower metabolism seems to promote correct folding of your
protein.

Ruth McMahon wrote:

> Hello,
> I am working with the pBAD bacterial his tag expression system from
> Invitrogen, and am trying to purify the his-tag protein. I have checked
> a number of methods to lyse the cells to release the protein, but in
> each case the vast majority of my protein stays in the pellet. I have
> used lysosyme, freeze thaw, sonication and NP-40 (NP-40 followed by
> freeze thaw seemed to work the best). I even tried 8M urea, even though
> I'd prefer to use native conditions. Does any one have any
> recommendations of how to generate a lysate which will contain the bulk
> of my protein?
> Thanks for any help,
>
> Ruth.
>
> --
>
> Ruth McMahon, Ph.D.,                                Tel: 353-1-706-2815
> Biotechnology Centre,                               Fax: 353-1-269-2016
> University College Dublin,
> Belfield,
> Dublin 4,
> Ireland.
>
> ---





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