[Protein-analysis] Re: Hints for protein export periplasmatic
clement at bio.mls.eng.osaka-u.ac.jp
Mon Sep 25 22:55:43 EST 2006
Assuming that your protein IS transported to the periplasmic space in
Salmonella, do you know which pathway does it utilize for transport? If it
is Sec, then I don't think you should not have any problems with the signal
sequences that are available with the commercial plasmids. Just remember to
exclude your native signal sequence (usually the first 14-22 amino acids)
when you use e coli's. In this case, lowering the IPTG concentration to
0.1 - 1 mM might work. Just adjust the overproduction time with the IPTG
concentration (this is largely trial and error). Lowering the overproduction
temperature to 30 C might also work. Do not lower the temperature too much
because the transport process needs ATP.
If it does not utilize Sec pathway for transport, then perhaps your e coli
does not have the right machinery for your protein to be transported OR for
it to stay transport-competent in the cytosol (if you use e coli's signal
sequence). If you protein has multiple cysteine residues, which may indicate
SS bonds, using e coli strain with oxidizing cytosol (and with overproduced
chaperones) might help. Some strains are commercially available.
If it is still not successful, do try various additives for renaturation.
Hope this helps.
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
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