Help! pET system not working

JG. Head bijgh at
Wed Jul 12 09:15:16 EST 1995

I'm having problems with my pET system.  I've successfully managed to 
ligate an insert coding for the 24kDa protein I want to express into 
pet15b.  I then transformed the plasmid into BL21(DE3)pLysS, added 1mM 
IPTG, and waited for over-expression.  Everything seemed to be working - 
nice over-expression of a protein of about the right size which purified 
out fairly easily on a His.Bind column (though it did elute at fairly low 
levels of imidazole).  I thought all this was too easy to be true, and it 
was.  When we sequenced the peptide it turned out to be chloroamphenicol 
acetyl transferase - a protein which is expressed by the pLysS plasmid.  
No sign of my protein at all, and inducible over-expression of an 
antibiotic-resistance protein (no sign of CAT if I don't add IPTG).  Not 
what I wanted!
Has anybody had any similar experiences or any ideas what may be going 
on?  Any suggestions would be very gratefully received,


Jared Head     at the Department of Biochemistry, University of Bristol
jared.head at

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