folding of cysteine-rich fusion peptide

Michael Morales mmorales at acpub.duke.edu
Tue Oct 15 11:45:03 EST 1996


I'm not sure if this is what you're after, but there is an E. coli mutant 
strain, AD494, that allows intercellular disulfide bond formation.  See 
Derman, et al (1993), Science vol 262:1744.  It can be purchased from
Novogen.  As far as protease problems go, check out the chapter by Beynon & 
Oliver in Methods in Molecular Biology Vol 59: Protein Purification Protocols
(1996; ed S Doonan, Humana Press).  In short, just because you add PMSF or any
other protease inhibitor, doesn't mean you still don't have a problem.

Hope this helps, good luck

Mike Morales

p.j. van santbrink (santbrin at CHEM.LEIDENUNIV.NL) wrote:
: 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



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