Re: [ccp4bb]: Culture in large scale

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

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Neratur K Lokanath wrote:

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> Dear All,
>
> Sorry for posting this non-CCP4 related question.
>
> I try to express the foreign protein in E. coli.
>
> When I tried small scale expression (below than 10 ml),
> protein can be solubly expressed. However, protein is
> completely insoluble in large preparation. Therefore, I
> think the best way might be culture the cells in 50 bottles
> of 10 ml culture (500 ml culture). Please send me some
> better suggestions.
>
> Thanks in advance,
>
> Sincerely,
> Lokanath
>
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Hi,

what temperature are you growing your E. coli's ?

Sometimes it helps to reduce the temperature to 30 or even 20 degree
celsius. I assume you're feeding them with LB medium. Is the pH adjusted to
7.2 ? Alternatively you might want to try out Terrific Broth, which is an
enriched LB medium with additional phosphat buffer. The cell yield is
approximately doubled in comparison to normal LB. By the way, what is the
yield of your protein in mg/ml culture roughly ?

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
Am Klopferspitz 18a                              http://www.biochem.mpg.de/
D-82152 Martinsried, Germany