Re: [ccp4bb] water soluble protein that needs detergent to be stable

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

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Subject: Re: water soluble protein that needs detergent to be stable
From: Daniel Picot Daniel {- dot -} Picot {- at -} IBPC {- dot -} FR
Date: 2007-08-29

I have not dealt myself with this type of protein, but Alex McPherson
tested the detergent beta-octylglucoside (at a concentration of 1.5 %,
i.e. above the cmc) for the crystallisation of soluble protein and tRNA
(1986, J.Biol.Chem 261:1969-75), the detergent did not hampered the
crystallisation, and even sometimes improved the crystal quality. It is
possible to somewhat control the detergent concentration: if the cmc is
high enough (something above 1 mM) you can dialyse the detergent,
otherwise you can bind the protein to a small affinity column and djust
the required concentration of detergent. I stop here because I find
always very difficult to extrapolate the effect of detergent from
membrane protein to soluble protein.
Daniel

Daniel Jin a écrit :
> Hi,
>
> I am working on a 60 kDa C. elegans protein that is predicted to be
> mostly alpha-helix. It is over-expressed in E.coli and the yield is
> about 1 mg/L of cell culture. The CD spec at 4 degree showed the
> presence of dominant alpha-helix. However, we don’t have any functional
> assay to confirm that it is folded correctly.
>
> It is over-expressed as a GST-fusion. We noticed that after cleavage of
> GST, it will easily precipitate if moved to room temperature (solution
> turns cloudy). Otherwise, it is OK at 4 degree. The CD temperature
> melting experiment showed a gradual change of signal, no sharp
> transition was observed. We later found out that including some
> detergent in the buffer will make it stay soluble at room temperature
> and showed as a dimer on SEC (4C or RT). Glycerol at 10% will help too
> but not as good as detergent.
>
> My concerns are, first this protein might not folded correctly, second,
> the presence of probably high concentration of detergent in the final
> sample will harm crystallization since the detergent will be
> co-concentrated with the protein. I am wondering whether anyone has deal
> with proteins like this before and their experience on improvement of
> the biochemical behavior and of course crystallization. Many thanks.
>
> Best,
> chen
>
>
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CCP4bb navigation

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

Previous message:
Subject: Resid for occupancy
From: Vu Thai thaivt {- at -} GMAIL {- dot -} COM
Date: 2007-08-28

Next message:
Subject: Re: The importance of USING our validation tools
From: James Stroud jstroud {- at -} MBI {- dot -} UCLA {- dot -} EDU
Date: 2007-08-29