recombinant protein expression problem

Laura Moen lmoen at
Thu Dec 17 15:11:46 EST 1998

Hi there everyone -
   I have been struggling with a difficult protein lately, and I would
appreciate input from anyone with an idea.  We have been attempting to
express a protein of approximately 100kD in size using a pET vector.  We
aren't getting good expression - can't see anything obvious on the gel
in crude extracts.  We ran an expression in the presence of 35S-met to
label our protein using rifampicin to shut off E. coli RNA polymerase
according to a protocol in the Red Book.  Our positive control worked
beautifully (another expression in the same pET vector, different
gene).  Our construct of interest gave us two things: a very faint band
of much higher than expected molecular weight, and a heavy band running
with the dye front.  Our negative controls gave faint bands running at
the dye front. My question is this: is the larger than expected band our
stuff and it is just getting degraded?  If so, why didn't I see any
laddering or smearing from the multiple degradation products?  Are we
getting readthrough from our stop codon which is generating the larger
product?  Or, is our stuff prematurely truncating for the most part,
hence the small stuff at the dye front?  We have sequenced the gene
several times, now, and I am confident there aren't any errors which
would cause an early stop.  However, we have yet to do an in vitro
transcription/translation - plan to do this later too.  Any input anyone
has, however, is very welcome.  Thanks in advance.  Laura Moen
LMoen at
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