E.coli lysis no enzyme damage

soenke behrends behrends at plexus.uke.uni-hamburg.de
Thu Mar 1 08:30:43 EST 2001


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 
Soenke











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