Q: fusion protein expressin

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Fri Dec 18 02:17:00 EST 1992


Jackson,
    We have been recently been using the pGEX system with 
great success.  We do use another strain; JM109.We have tried
 other strains without luck (DH5 alpha).   We also do not do the column thing.
 
We just put in 50 to 100 ul of a 50% slurry into a 1 ml extract and batch it. 
 
We have also found that glutathione
only removes about 1/2 of the protein that is stuck, as assayed
by cracking the beads with SDS PAGE loading buffer.  
This sacrifices beads but it is one way to assay if things
 are working in light of the inability of glutathione to
elute all the bound protein.  We have used 15 to 50 mM glutathione
 with little increase % eluted.(Still have about 1/2 stuck to the beads
if crack with SDS loading buffer).  But even with this limitation we  
enough protein to see big fat band on a coomassie stained gel.  One thing
you did not mention was the control.  Do you get GST if you just use
non-inserted vector?  This was the first experiment we did and it works
alot better than any fusion we have tried.  We also notice that if we grow 
the bacteria containing the fusion at 25 or 30 degrees rather than 37 degrees
we get more of the full length protein.  In this case we still do a 2 to 4 
hour 
induction and still manage to get loads of fusion protein.
My guess is that if your column is very large, lets say 5 mls, then your
fusion is probably getting hung up because the glutathione does seem to 
be the best elution agent.  Try the BATCHing trick with a small amount of 
matrix and crack it with SDS buffer.  I'm sure that if you start with GST
and get this working the rest will fall into place.

Dan Gietz
U of Manitoba
Winnipeg, Manitoba    were its -26 degree C outside now!
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA 




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