Dear fellow netters
Using the pGEX system from Pharmacia I'am trying to
isolate a eucaryotic protein with many cysteines.
I've succeeded in getting most of the fusion protein
in the soluble fraction of my cell lysate (after growing
the bugs for 2 hours at 370C I induce with 0,5 mM
IPTG at 300C for 2 hours in the presence of 2.5 mM
Betaine and 0,1 M Sorbitol in normal LB-medium). I'am also
able to purify the fusion protein with the glutathion beads.
My questions are:
- is it true that with this isolation procedure (using pGEX
vector system and the above mentioned conditions) the SS bonds
are not formed?
- if this is the case (and I think so because I've recently read
that these bonds are only formed in the periplasm), is there a
way to fold the protein after isolation in a CORRECT manner (
this is essential because I have to perform binding studies with
the fusion protein)?
- when I bring the fusion protein on SDS PAGE gel (add part of lysate
to loading buffer - 0,1 % SDS + mercaptoethanol-, heat 5' 950C) the
location of the band is to low (at 35kD and should be at 45 kD); is
there a explanation (I use PMSF troughout the isolation)?
I hope someone out there in cyberspace and beyond can help me.
Peter van Santbrink
Division of Biopharmaceutics
University of Leiden