Date: 30 September 2011 15:49
Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae)
Thanks,
Pat
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Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
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From: Ed Pozharski
Wait, isn't Rosetta optimized for mammalian genes, not yeast (but maybe
codon bias in yeast matches that)?
A sideways suggestion would be to express in yeast. Extra bonus - it
smells like beer :)
--
"Hurry up before we all come back to our senses!"
Julian, King of Lemurs
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From: Craig A. Bingman
Rosetta strains carry a plasmid that supplies several tRNAs that match codons that are rare in E. coli. This is quite different than being explicitly optimized to express mammalian proteins. We (the Center for Eukaryotic Structural Genomics, a PSI-1 and PSI-2 center) used strains with the same tRNA plasmid (either pRARE or pRARE2) to express proteins from yeast, Arabidopsis, some thermophilic eukaryotes, mouse, frog, human and zebrafish proteins. It seemed to work just fine for all of them.
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From: Tim Keys
We codon optimised a poorly expressed gene from neisseria meningitides based on a codon usage table derived from the Welch (etal, 2009) paper below. The optimisation is specifically for overexpression in BL21 (DE3). The optimised gene increased protein expression by at least a factor of 10, and changed (somewhat reduced) the degradation pattern we observed. Unfortunately it didn't do anything to improve the folding (ie. we ended up with lots of half-folded, semi-soluble protein).
With other neisserial derived proteins we have had an almost undetectable effect.
You can't win 'em all.
Cheers,
Tim
Design Parameters to Control Synthetic Gene Expression in Escherichia coli
Welch et al, PlosONE 2009
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From: Ed Pozharski
Sure - what I meant was optimized codon usage. I guess to answer the
original question, one could use rare codon calculator
http://nihserver.mbi.ucla.edu/RACC/
and then see if the addition of tRNAs for AGA, AGG, AUA, CUA, GGA, CCC,
and CGG in Rosetta2 would solve the problem for a particular sequence.
(It's likely that Pat has done that already). I don't know if those
cover all the rare codons in E.coli or if there is something in yeast
sequence that is still missing and thus adjusting the codon usage will
help. In theory, if the rare codons are all covered by Rosetta's extra
tRNAs, codon optimization should not make any difference.
--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs
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From: Dima Klenchin
In practice it does because frequently it's not codon optimization per se but changing local mRNA structure.
- Dima
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From: Segelke, Brent W.
To me, the key question would seem to be, if I can't win them all, how many more do I win if I go to the trouble?
Brent
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From: Anastassis Perrakis <a.perrakis@nki.nl>
Date: 30 September 2011 18:07
To: CCP4BB@jiscmail.ac.uk
Sent from my iPad
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From: Segelke, Brent W.
Is there a general consensus that this is true? I've heard exactly the opposite, i.e., that codon optimization rarely gives you dramatically improved yields of soluble protein. Are there any published studies on this topic? This seems like something that might come out of one of the SG centers.
Brent
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From: Torres-Larios Alfredo
We recently published something really "old-related" about it:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0021035
My two cents. Best, Alfredo.
Alfredo Torres-Larios, PhD
Assistant Professor
Instituto de Fisiologia Celular, UNAM
Mexico
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From: Luca Jovine
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