Depending on your exact question, I can see the following possible
problems:
- a stop codon has been introduced after the GST, either by a cloning
error or spontaneous mutation (if the fusion protein is toxic, these
sponateous mutations may be relatively frequent). Are you using
glycerol stocks for your starter culture? Try using a fresh
transformation from the day before to minimise introduction of
spontaneous mutations. In fact, I never use glycerol stocks, and
always use fresh transformations, after finding out this problem the
hard way and losing valuable time.
- the fusion protein is unfolded/toxic/otherwise problematic, and
proteases chew it away during expression. Try growing the culture, and
also the starter culture, also at 20 ºC also before induction. In
combination, also try induction with low concentrations of IPTG (0.01
mM or less) to ensure plasmid-bearing cells stay viable and are not
outgrown by others.
- or, if expression of the fusion protein is ok, but you get only GST
and little of the eukaryotic fusion partner upon protease cleavage, it
may be that the partner is poorly folded and only kept in solution by
the GST. Here, a possible solution would be an eukaryotic expression
system.
Mark J. van Raaij
Dpto de Bioquímica, Facultad de Farmacia
Universidad de Santiago
15782 Santiago de Compostela
Spain
http://web.usc.es/~vanraaij/
On 17 Oct 2008, at 14:39, Putcha, Balananda Dhurjati Kumar wrote:
Dear CCP4ers,
I have a GST fusion protein (eukaryotic). When I express it in
E.coli BL21 (DE3) or RIPL cells, however, I mostly get GST and very
little fusion protein. I get very little "leaky expression". I
normally grow my cells at 37C in LB or 2X YT and after induction at
20C for 16-18hrs. I also have tried growing at 37C for 2hrs (after
induction) with little change. Is there a trick to solving this
problem? Thank you for your thoughts
Kumar
