Re: [ccp4bb] bacillus protein expression

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Rejected posting to CCP4BB@JISCMAIL.AC.UK
From: "William G {- dot -} Scott" wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2010-04-15

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Subject: Re: Rejected posting to CCP4BB@JISCMAIL.AC.UK
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2010-04-15

Subject: Re: bacillus protein expression
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2010-04-15

1. look up Bacillus megaterium expression system (commercial, pretty
cheap & relatively easy). It's easier to use than some of the
integration-based B. subtilis systems. Or you could ask around for a
suitable B.sut. shuttle vector as an alternative.

2. Bacillus codon set is heavily biased towards AT-richness and even
in the codon+ may not be expressed well - there may also be mRNA
issues (stability). It's not hard to make synthetic genes these days -
if you're desperate to continue using E. coli for this work you can
make a synthetic codon-optimized version specific to the host.

Cheers,

Artem

On Wed, Apr 14, 2010 at 2:30 PM, Amit Kumar wrote:
> Hi all,
>
> Sorry for a non-ccp4 query.
>
> I will highly appreciate your comments on the expression:
>
> I am trying to express HIS tagged ~60 kDa cytosolic protein from Bacillus in
> E.coli pET expression system. I tried codon+ strain, time of expression
> and various temperatures ( 18, 25, 30, 37C) for the expression. Neither
> full-length nor truncated form of the protein was found to be expressing
> (also checked by western blot). What other parameters should I explore to
> get the expression in Escherichia coli?
>
> Thank you very much for your comments.
>
> Amit K.
>

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Rejected posting to CCP4BB@JISCMAIL.AC.UK
From: "William G {- dot -} Scott" wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2010-04-15

Next message:
Subject: Re: Rejected posting to CCP4BB@JISCMAIL.AC.UK
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2010-04-15