IUBio

Inclusion bodies

Frank Fürst ffrank at rz.uni-potsdam.de
Mon Aug 2 09:01:52 EST 1999


Dr Lester Davids wrote:
> 
> Hi All,
> 
> Looking for advice as to how to PREVENT proteins from getting into these
> damn bacterial inclusion bodies! It seems out native proteins are not
> but our mutant proteins are. Any suggestions out there?
> 
Well, as I said in reply to your first posting: (Destabilizing)
Mutations are a frequent reason for aggregation. In particular, there
are two possibilities:

1. As described earlier, there is a folding intermediate that is
susceptible to aggregation, either like this:

a)
U ---> I ---> F
       |
       ---> Aggregates,

where U denotes unfolded and F native folded Protein, or like this:
b)
           /--> F
          /
U ---> I-on <---> I-off ---> Aggregates 
            
where <---> denotes an equilibrium between the on-pathway intermediate
I-on and the off-pathway intermediate I-off
The effect of the mutation could be 

(in a and b) to slow down folding from I (I-on, respectively) to F, so
that I (I-on + I-off) accumulates and the aggregation reaction (n I
---> In) with reaction order n gets faster than first-order folding to
F, or 

(in b) (which I think occurs more frequently) a shift in equilibrium
between I-on and I-off. Thus the concentration of I-off increases, and
aggregation occurs.

In either two of these cases, my tips could help (decreasing
expression level, or temperature, or coexpressing chaperones, or
solubilising IBs and refolding). 

2. The second possibility is that the mutations affect the stability
of the (native structure of the) protein enough to prevent it from
folding at all; what was the native structure in the wild-type is now
energetically unfavorable compared to a compact intermediate with at
least the tertiary structure beeing flexible (and this intermediate,
in turn, is less stable than the aggregates formed out of it).

If this is the case, you won't rescue your protein (but you've learned
something on structural important residues...)

If you only have single point mutations that do not involve Cys making
disulfide bridges, the second case is very unlikely. But if you have,
e.g., amber mutations, you should also think of that.

hope this helps, Frank
-- 
Frank Fuerst, Institute of Biochemistry of Potsdam University
Im Biotechnologiepark, D-14943 Luckenwalde
Tel.: +49-3371-681334;   Fax: +49-3371-681339

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