Hints for protein export periplasmatic space during expression
Hi there,
I would appreciate all ur posts here about hints u use or know how to
determine such conditions in that the bacteria (I'm thinking about
particulary 3 strains we have in our labs E.coli C600, E. coli
DH5alpha, and BL21 with lambdaDE3) to produce soluble protein (I mean
exported to the periplasm). We use different plasmids - all of them
have specific signal sequence either specific for strain E.coli or for
our protein (which comes from Salmonella Enteritidis strain). We search
for optimal conditions for expression the protein and spedition to the
periplasm, howether in all cases protein in 99% appear as inclusion
bodies. Renaturation wa unsuccesful.
So we heard about
1 inducting the culture by addition IPTG very early (when OD600 = ~0,2)
do u agree it is good idea?
2. lowering the temperature after induction to 27C deg and let it grow
in the presence of IPTG for 12 16h.
But maybe there are more efficient tricks?
I would appreciate all opinions
Bres
