Re: [ccp4bb]: Expression of archeal proteins

[Juergen Bosch <jbosch@biochem.mpg.de>]

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Narayanan Manoj wrote:

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> Dear all,
>           I am trying to express an archeal protein in Ecoli. Can
> someone name some references that lists commonly used expression and
> purification protocols in such cases.
> Thanks a lot
>
> manoj
>
> Narayanan Manoj,
> 372, Baker Lab,
> Cornell University, Ithaca, NY 14853-1301

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)

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

Assuming your archeon has a high temperature optimum you can use heat
shock as a first purification step with a diluted crude extract (10 ml /
g wetweight).

Good luck

Juergen


--
Juergen Bosch                                    Tel -49 -89 8578 2648
Max Planck Institut fuer Biochemie               Fax -49 -89 8578 2641
Abt. Molekulare Strukturbiologie                 bosch@biochem.mpg.de
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