E.coli lysis no enzyme damage
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Thu Mar 1 12:34:52 EST 2001
behrends at plexus.uke.uni-hamburg.de wrote:
: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.
Your enzyme either does not fold properly in E.coli
(even if some of it is soluble) or requires some
posttranslational modification for activity. In either
case, the problem is extremely unlikely to be caused
by too harsh sonication - in this case you would be
killing say 50 or 80% of total activity, but never
: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?
If you are getting extreme viscosity in the solution, then
it is sufficient. Usually, 1 mg/ml is used, though.
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