Help! pET system not working

JG. Head bijgh at zeus.bris.ac.uk
Wed Jul 12 09:15:16 EST 1995


Hi,
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,
Thanks,

Jared

--
Jared Head     at the Department of Biochemistry, University of Bristol
jared.head at bristol.ac.uk



More information about the Methods mailing list