Trond Erik Vee Aune
teva at online.no
Mon Mar 11 06:06:36 EST 2002
Chandra shekhar Boosani wrote:
> Dear all,
> could you please suggest me for how to enhance protein translation.
When trying to express proteins getting efficient translation is often
the bottleneck. Here are some suggestions:
1) Sometimes it can be fruitful to optimize the codons for the organism
you are using for the expression.
2) Ensure everything is ok at the 3'-end of your mRNA so it doesn't gets
3) Enhance the efficiency of the translation initiation region in ways
3a) Make sure you use an optimal SD-region for your organism.
3b) Remove sub-optimal secondary folding of your mRNA around the
translation initiation region (TIR). One way is to use a native gene or
cistron upstream of your gene of interest. A native gene will probably
not cause negative secondary structure and will channel ribosomes down
towards the gene of interest. The positive effect of this can often be
seen when using signal peptides in E.coli fused upstream of your gene.
Of course using signal peptides is only a possibility when you want to
translocate your protein to the membrane. If you must have your protein
located in the cytosol I would suggest trying to use re-initiation
strategies: clone a small part of a native gene you know is translated
with high efficiency upstream of your gene, then make stop codon(s)after
this truncated gene and make a new TIR between this truncated gene and
your gene of interest. In theory the ribosomes thats binds to the TIR
upstream of the truncated gene should now be channeled towards the
stop-codon(s) and the new TIR, this channeling and steady influx of
ribosomes should overcome secondary structures around the second TIR
caused by your gene.
> Thanks in advance
> yours sincerely
> Chandra shekhar Boosani
> Ph.D Plant sciences
> University of Hyderabad
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