folding of cysteine-rich fusion peptide

[p.j. van santbrink]

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
The Netherlands