C-termianl his tag
hubahopp at gmx.de
Wed May 14 18:50:05 EST 2003
there's no need for a program or software tool.
if you want to express it in e. coli, use the rbs of e coli, of course (ec
won't talk caulobacter language if there's any difference at all in respect
to rbs). the n terminal his tag you probaly don't want, so re-decide your
decision regarding the vector choice (ec rbs and nterm his tag then
something then your gene is likely to be fatal concerning your experiment).
so basically, select a vector that has a c terminal his tag or simply
design 2 primers (n terminal with ec rbs and atg an c terminal wis 6his and
tga=stop). backbone shouldn't be too critical, but finally depends on your
expression host (promoter/plasmid copy number-ori!!!).
rbs is not just yes/no, but rather fuzzy in term of better/worse, so always
choose the best you can get for high expression. But remember, it's a
living system, not a computer, so expect false positives/negatives to happen.
all the best
PS -important- to all bionetters. Wo is currently unemployed and looking
for a new job (postdoc or higher level anywhere in this world (why not
mars?) with relation to molecular biology/cell biology/taylor made
proteins/fermentation/directed molecular evolution. Please drop me an email
for discussion if you have a position to offer!
At 16:55 14.05.2003 -0400, mj wrote:
>I want to C-terminally His tag a protein from Caulobacter (a gram-negative
>bacteria), and I am thinking about using pET-28a vector from Novagen. But
>this vector carries both an N-terminal His tag and an C-terminal one. If I
>clone my gene in frame with the C-terminal His tag, do I need to clone the
>ribosome binding site of that gene from the Caulobacter (but I want to
>express it in E.coli)?? The order of the sequence in that vector is: rbs--N
>terminal his tag--restriction site--C termianl his tag
>And do I need to remove the N-terminal his tag??
>If I need to clone the rbs from the Caulobacter, is there any software or
>tool that I can use to search for the ribosome binding site of a gene?? I
>don't know the exact sequence of rbs in Caulobacter, and I think it's
>different from E.coli
>Help will be much appreciated!! Thanks!!
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