s330477 at student.uq.edu.au
Wed Apr 10 01:13:24 EST 2002
Most EGF-like domains contain 3 intra-disulfide bonds.
The cytosolic environment of E.coli is reducing and no disulfide bond
When you lyse the cells, you release your recombinant protein into an
oxidative environment where disulfides will form "randomly".
To form native disulfide bonds in vitro, you can use a mixture of 3mM
reduced and 0.3mM oxidised glutathione at pH 8.3 to allow thiol-disulfide
exchange (generally 24 h at 4° is sufficient to reach equilibrium). You can
introduce the GSH/GSSG mix into your dialysis buffer, or add it to purified
protein. Additionally, some EGF-like domains require calcium for folding.
In this case, you need to include Ca (eg 2.5mM CaCl2) in the refolding
buffer. You can then isolate the correctly folded isomer by RP-HPLC.
This method has worked well for me.
Hope this helps.
John Hwang ;)
"Lorenza" <lorenza at dba.unito.it> wrote in message
news:20020405175100.13076.qmail at ww02.jatek.com...
> I have produced a recombinant NRG egf-like domain, fused with His tag, by
> E.Coli. I have extracted it from inclusion body using UREA.Then I have
> purified it by NicKel colum and dialyzed.
> Is the dialysis sufficient to renature my protein?
> What can I do to optimize renaturation so that I can be sure to have my
> protein in an active form? I need the EGF-like domain binds to his
> Thank you very much!
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