Inclusion bodies (aaaaahhhhhhh!!!!)

Peter Gegenheimer pgegen at DawnTreader.bio.ukans.edu
Tue Jun 27 11:16:26 EST 1995


In <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 
>
There are several things you can try. The first is an old standyby; the 
second comes from a paper from Bob Fletterick's lab (I think in
Protein Engineering)-- we haven't tried this yet.

First, grow the cells at 15 to 20 degrees C -- (you might be able to shift 
them from 30deg to 20 deg just before induction).  THis may work, but not 
always.Second, induce expression to a very low degree -- for example, use 
about 1/10 to 1/20-th of the normal amount of IPTG, or 10 to 40 micromolar. 
Let the cells "induce" overnight or longer, and you can get large amounts of 
csoluble protein. Fletterick's lab used this to get 70-100% soluble eukaryotic
glycogen phosphorylase (97kDa) expressed as active dimers in E. coli. The two 
expression vectors were a pET vector, and a plasmid with dual trc (or tac?) 
promoters. (rRNA promoters are also good, as they contain an antiterminator 
activation site.)

Another post suggested co-expressing groEL. We tried this with no success; the
groE experts say this works sometimes but not always. (The mass of groEL you'd
need to make even catalytic amounts of the active 14-mer is pretty 
substantial).

In favor of inclusion bodies, they generally are >90-95% pure target protein, 
so purification consists of washing the pellet thoroughly! Refolding may be 
attempted for larger proteins according to Chen et al., FEBS Lett 298, 69-73 
(1992) and Chen et al, Biochemistry 270, 1-9 (1995)(tentative - in press for
Aug.).

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