Ox. reagent for proteins

bipin dalmia dalmiabk at phibred.com
Fri Dec 16 11:38:55 EST 1994


In article <9412142220.AA04574 at umailsrv1.UMD.EDU> lw75,
Lynne_A_WHITEHEAD at UMAIL.UMD.EDU writes:
>I was wondering if anyone knows of a way to form disulfide bonds in a
>protein in vitro.  To explain alittle, I am trying to clone a hemolysin
>protein into E.coli using pMAL.  The 2 pMAL vectors will either express
the
>resulting fusion protein (hemolysin fused to a maltose binding protein)
in
>the cytoplasm or in the periplasmic space.  Sometimes the fusion proteins
>are too large to be transported to the periplasmic space, so I may have
>problems with this.  Also, the literature tells me that the
cytoplasmically
>expressed fusion protein will not allow for disulfide bonds to be formed.
>This is a problem since there are essential disulfide bonds in my
protein's
>structure for it's activity.  First, does anyone know why or what keeps
>disulfides from being formed in the cytoplasm (pH?, ect.)? and are there
any
>oxidizing reagents (or anything) that can be used to produce the
disulfide
>bonds (in vitro) in my proteins once it's been isolated?  Or will this
type
>of manipulation of the protein dammage it or not  produce the correct 3D
>configuration as it would be formed in vivo?

most proteins that require disulfide bonds form the correct ones when the
e. coli cells are lysed, as soon as the protein comes into contact with
air (dissolved). to avoid non-specific disulfide bond formation upon
lysis, add ca. 10 mM BME (or 1 mM DTT) to the lysis buffer. in case you
do want the disulfides to form in vivo (in the e. coli cell), ask Jon
Beckwith at harvard medical school to send you a mutated e. coli that
lacks the thioredoxin reductase activity. he has shown that this activity
is responsible for the lack of disulfide bond formation in the e. coli
cytoplasm. the mutated e. coli allows for disulfide bond formation in the
cytoplasm. dr. beckwith sends the e. coli free to academic researchers
but charges $5000 to us industrial ones :(

bip



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