Re: [ccp4bb] bacillus protein expression

- Protein crystallography

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

Previous message:
Subject: Re: X-Ray films
From: Brad Bennett bradbennett76 {- at -} GMAIL {- dot -} COM
Date: 2010-04-15

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Subject: composite omit map in CCP4
From: weikai {- at -} CRYSTAL {- dot -} HARVARD {- dot -} EDU weikai {- at -} CRYSTAL {- dot -} HARVARD {- dot -} EDU
Date: 2010-04-15

Subject: Re: bacillus protein expression
From: Brad Bennett bradbennett76 {- at -} GMAIL {- dot -} COM
Date: 2010-04-15

Hi Amit-
We used a well-expressing soluble protein fusion (SUMO) at the N-term of a
couple of B. anthracis proteins that we were trying to produce in E. coli,
which improved the soluble yield of both targets tremendously. The
disadvantage of a fusion is of course you produce a "non-native" protein-
upon cleaving away the fusion (e.g. with thrombin or TEV), you will probably
retain one or more exogenous residues. We picked SUMO because after cleaving
with Ulp1 (SUMO protease) you produce a native protein with no additional
residues from the fusion. If you want more details about this and things we
tried to boost expression, please feel free to contact me off-list.

Best-
Brad

On Wed, Apr 14, 2010 at 3:30 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.
>

CCP4bb navigation

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

Previous message:
Subject: Re: X-Ray films
From: Brad Bennett bradbennett76 {- at -} GMAIL {- dot -} COM
Date: 2010-04-15

Next message:
Subject: composite omit map in CCP4
From: weikai {- at -} CRYSTAL {- dot -} HARVARD {- dot -} EDU weikai {- at -} CRYSTAL {- dot -} HARVARD {- dot -} EDU
Date: 2010-04-15