Cloned, but not expressed.

Paul Jasper pjasper at orion.luc.edu
Sun Aug 29 16:28:40 EST 1999



Andrew Leung wrote:

> Hi,
>
>     I have cloned my favorite gene into a fusion gfp vector. But it didn't
> express.
>
>     My question is that:
>
> Is translation always start at the first AUG codon the complex first
> encounters? If yes, I don't know why my gfp didn't come up.
>

>>  Translation doesn't have to start on the first AUG... it depends whether or
not it is in the context of proper Kozak (sp?) consensus sequence.. or more or
less whether it is in the context of a ribosomal binding site.


>
> Is there any sequence in the transcript (which upstream to any AUG) that can
> determine whether an AUG could be the starting site for the translation?
>

>> yes.  I think the ribosomal binding site is part of the 5' untranslated
region (I'll let the newsgroup correct me if I'm wrong... :-)


  I would suggest that your problem may be in the plasmid construction
itself...with a more obvious problem.  Are you sure you cloned your gene in
frame with the GFP gene??  Are you sure your gene doesn't have a stop codon in
the ORF ?.. are you expressing the plasmid in a cell type which would transcribe
from your promoter?


good luck,

         paul


--
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\  \ / \  \ / \  \ / \  \ /    Paul Jasper     \ / \  \ / \  \ / \  \ /
/\  \  /\  \  /\  \  /\   Micro/Immuno - Loyola U. /\  \  /\  \  /\  \
  \__\/  \__\/  \__\/  \__pjasper at orion.luc.edu__\/  \__\/  \__\/  \__\/





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