E.coli lysis no enzyme damage
ekhatipo at NOSPAMmidway.uchicago.edu
Thu Mar 1 12:40:58 EST 2001
I would freeze induced E.coli cell pellet at -80 to store, and when needed
thaw the pellet, add buffer with protease inhibitors, lysozyme and DNAse I
(~<10U/ml), freeze thaw the suspension couple of times. After
freeze/thawing, I would sonicate the lysates real quick (1 x 15 sec; that
would kill DNAse I by the way), and spin down debris. Make sure no foam is
formed during sonication.
"soenke behrends" <behrends at plexus.uke.uni-hamburg.de> wrote in message
news:97lisd$ssd$1 at rzsun03.rrz.uni-hamburg.de...
> Dear netters,
> we are getting nice soluble expression of a
> 80 kDa His-Tagged enzyme in E. coli. (about
> 10 % soluble and 90 % inclusion bodies).
> Though we can measure activity when the protein
> is expressed in Sf9 cells, we were not success-
> ful in E. coli so far. It is not clear whether
> that is due to the differences between eukaryotic
> versus prokaryotic expression or whether
> our Coli-lysis by sonification is too
> harsh. With Sf9 cells we use a high pressure
> nitrogen bomb, but we do not want to use bacteria
> in that bomb.
> Is digestion with lysozyme 0.1 mg / ml for 30 min on
> ice sufficient for lysis of coli? And is is regarded
> as a method where expressed proteins / enzymes are
> left more or less intact. Is there a better method?
> Thanks for your time and help
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