Toxic protein for E. coli (M15)
junk at hgmp.mrc.ac.uk
Thu Sep 5 08:08:20 EST 2002
Historians believe that in newspost <3D775480.A631B11E at abdn.ac.uk> on
Thu, 5 Sep 2002, Bertrand Collet <b.collet at abdn.ac.uk> penned the
following literary masterpiece:
>> Do you get an extra protein band produced for your protein in your pQE30
>> clone as opposed to pQE30 alone?
>I have never seen any extra band so far. I lyse the bacteria by lysosyme
>+ sonication and checked the soluble fraction on SDS-PAGE.
>Is it possble that the protein is expressed and then "disappear" from
>the soluble fraction (inclusion bodies)
Inclusion bodies won't be soluble fraction. They will come down in the
debris pellet after sonication.
> i.e. 4 hours is too long ?
Maybe but that is for checking
>I hope to find out more on a time course of expression (at this very
>moment: 0, 30 min, 1, 2, 3, 4 and 5 hours).
It could be lethal even at a level too low to see on an SDS PAGE. It
could be rate limited based on codon usage and umpteen other things.
Can you do an assay for the protein itself?
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
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