help lysing E. coli to purify inclusion bodies

[Peter Gegenheimer]

In <16FE48840S86.FJVAND00 at ukcc.uky.edu>, FJVAND00 at ukcc.uky.edu writes:
>In article <Cs4CCt.595 at ucdavis.edu>
>jfh <jfhess at ucdavis.edu> writes:
>>I (we) are making mamalian proteins in ecoli using pT7 and BL21(DE3).
>>
>>I've had success with the system but my lysis is always a crap shoot.
>>Sometimes lysis occurs easily sometimes not.  I have been following the
>>methods in enzymology, nagai and thorsen protocol, using lysozyme,
>>followed by deoxycholic, NP40, and DNAse 1.
>>What do other people do?  Post some suggestions here or email me.
> 
>A very easy solution is to switch to BL21(DE3)pLysS, which lyses automatically
>upon freeze/thaw.  I thaw in a buffer containing 0.1% Triton X-100, but its
>probably not at all necessary.  A little bit of sonication still helps to
>reduce the viscosity of the lysate.

I second the above. Our lab has used BL21(DE3)/pLysS for several years now. 
the freeze-thaw "autolysis" works every time.

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