Ox. reagent for proteins
Lynne_A_WHITEHEAD at UMAIL.UMD.EDU
Wed Dec 14 17:26:35 EST 1994
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?
Thanks for any help!
U. of Maryland
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