help lysing E. coli to purify inclusion bodies

FJVAND00 at ukcc.uky.edu FJVAND00 at ukcc.uky.edu
Wed Jun 29 08:41:14 EST 1994


In article <Cs4CCt.595 at ucdavis.edu>
jfh <jfhess at ucdavis.edu> writes:
 
>
>Howdy all,
>
>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.
 
Good luck,
 
Frank van de Loo
USDA/ARS
Lexington, KY.



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