GST-fusion yield 50mg/0.5 lt culture?

[Cassie Conley]

In article <dmthomas-1010961457290001 at rablab.biochem.uci.edu>,
dmthomas at orion.oac.uci.edu (Didier Thomas) wrote:

> Induction should be started on an exponentially growing culture (OD ~ .5
> is OK) and should last at least 3 hrs.
> 
> Temperature : 30 or 37 C depending on your protein.

   I've got a toxic protein I express using pET16 in BL21(DE3)pLysS cells,
and my best overexpressed protein/bacterial protein ratio occurrs after
45min-1hr induction @ 25C using 1mM IPTG, at a culture density (eyeballed)
of ~1/2max -- probably OD ~1 (I inoculate directly from a glycerol stock
into LB+2%glucose to give the bugs less chance to mutate my plasmid --
usually grow 4-6hr).  Much longer inductions, no matter what the density,
and I see bacterial protein expression swamping out my protein.  I suspect
enough bugs are killing the plasmid so that after ~2 generations most of
the total protein is made in non-expressing bugs...

                                       -- Cassie Conley
                                          cassie at scripps.edu
                                          Cell Biology, MB24
                                          The Scripps Research Institute