Inclusion bodies (aaaaahhhhhhh!!!!)

wegloehner at mpimg-berlin-dahlem.mpg.de wegloehner at mpimg-berlin-dahlem.mpg.de
Mon Jun 26 12:12:38 EST 1995


In article <3sju4d$bbv at violet.csv.warwick.ac.uk>, jl at dna.bio.warwick.ac.uk writes:
> I am trying to express a monooxygenase in different bacterial strains
> including E. coli.  Although expression from the T7 promoter
> is seen, the protein seems to aggregate as inclusion bodies and is
> inactive.
> 
> Many methods such as fusion proteins and solubilisation of these
> inclusion bodies have been reported and are to be attempted.
> 
> Can I hear your ideas and views on getting over this problem? 
> 
> Thanks for your help!
> 
> John Lloyd
> Biological Sciences
> University of Warwick
> UK
> 
> JL at dna.bio.warwick.ac.uk 

Welcome to the world of bacterial protein expression!!!!!

ok, jokes of now. There are several things you can try to overcome the inclusion
body problem. First if your protein is a small prokaryotic protein your chance to
get it correctly refolded after resolubilization is quite good. In this case
inclusion bodies can be an advantage
If your protein on the other hand is eukaryotic and larger than approx. 40 kDa 
- I would say forget about it.
Ok now to your system. You can try the following things (separated or combined)

1. decrease the incubation temp after induction. Grow your bugs at 37 degrees
up to the induction OD - then cool them down to 30, 25, or 20 degrees and induce.

2. grow your bugs under osmotic stress. There is a paper by Blackwell and Horgan
(1991) which describes this method (FEBS Lett. 295, 10-12).

3. use a expression system which secretes your protein into the periplasmic space
of e. coli. We had success using this system when expressing ribosomal proteins
in e.coli.

4. co-overexpress a chaperon like groEL. Helps to fold your protein correctly. Or
if your protein needs any cofactor to work (specific ions or other components)
its a good idea to add this stuff into the media.

ok can't think of more at the moment - but have a look into Current protokols -
there are for sure some more hints about inclusion bodies and expression.
hope this helps

wolf



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