Re: [ccp4bb]: Expression of archeal proteins

[Anastassis Perrakis <perrakis@nki.nl>]

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hi (again)-

On a related issue: you can also of course back-translate and make the 
synthetic gene with E.coli codons. Any clues as of how much this costs 
these days and who does it ? I got one company for 3.50 Euro per bp. Too 
expensive for my taste.

 
> I assume you have checked your sequence for rare codons (RIL) ?
> 
> If this is not the case you should find out how many rare codons are
> present  in your sequence before cloning your gene into E. coli.
> A high content of rare codons >5% will reduce the yield of your protein
> significantly in E.coli (in my case 10% rare codons; 50 fold increase of
> my protein between normal BL21 and Codon Plus Cells)
> 

moreover a rare codon can result to half of your protein to be C-term 
truncated and happily sticking to N-agarose - in case of N-term tags ...
We need a good protease for removing C-term tags .. all I have seen cut 
close to the C-term of the linkers and thus would leave too many 
residues if tried with C-term tags ... Anything like that out there ?

> I can recommend the following strain from Stratagene BL21 Codon Plus
> RIL. You also might want to check the Rosetta E. coli strain.
> 

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