I was just wondering whether anybody out there has any experience
with expressing fairly short (70aa) eucaryotic polypeptides as fusions
of maltose binding protein (MBP) using the pMAL-C2 vector system (NEB).
I'd particularly like to know which E.coli hosts offer good levels of
stability against proteolysis without overly compromising on yield (do
Lon-deficient strains make a huge difference? I'm currently using my usual
cloning strain, XL1-Blue MRF' which gives excellent overexpression but
in vivo proteolysis looks to be quite severe...). Also,has anybody
produced modestly sized fusions that refuse to bind effectively to the
amylose affinity column? I think I have....
Any advice greatfully appreciated,