Bacterial expression with secretion

bioc07 at otago.ac.nz bioc07 at otago.ac.nz
Wed Nov 4 17:41:21 EST 1992


In article <1992Nov2.164053.1 at mscf.med.upenn.edu>, kaya at mscf.med.upenn.edu writes:
> I am currently expressing a fusion protein in BL21.  The vector I am using in 
> pEZZ-18 (Pharmacia).  From all available data, I seem to have extremely large 
> quanitities of fusion protein (almost too high).
> 
> My problem is this - when I try to cleave the fusion protein with cyanogen 
> bromide cleavage, the protein is no cleaving.  
> 
> I have done amino acid analysis 
> and can pick up the methionine (only one) in the fusion protein, but amino acid 
> analysis of post cleavage material also indicates the presence of methionine 
> (which should at this point be a homoserine or homolactone).
> 
> My question is, is there anybody else who is working in this system 
> (successfully or not) who would be willing to share experiences?  Or is there 
> anybody who works with bacterial expression vectors which secrete product
> to either the periplasmic space or into media (pRIT5, for example) who would be 
> willing to share the expertise? 
> 
> 
> Thanks,
> 
> angie.


Cyanogen bromide sometimes requires denaturation of the protein before cleavage
will occur. Typically 70% formic or trifluoroacetic acid can help although some
proteins are rather acid-labile. Some batches of formic acid seem to be worse
than others at causing non-specific cleavage, something which may be related to
dimerization and polymerization of the formic acid. Alternatively, you could
try heating your protein in 6M guanidinium HCl at pH7.5 to 65C for 10 minutes.

Sadly, all these things will cause almost complete denaturation of your protein
which may well be something you don't want to do. It might be possible to find
milder conditions where the methionine is cleaved but the protein is not
completely denatured. 

Cheers,

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