Refolding denatured protein

Nikolai Chitaev nikolaic at VISAR.WUSTL.EDU
Fri Feb 23 14:03:58 EST 1996


> 
> To: methods at net.bio.net
> 
> From: lpss at unixg.ubc.ca (Alex Chang)
> 
> Subject: Refolding denatured protein
> 
> Date: 22 Feb 1996 22:41:38 GMT
> 
> Hi, everyone:
> 
> I am expressing a single-chain antibody now, using an E. coli expression
> system (pET19b, Novagen). I have the protein expressed as show by
> SDS-PAGE, and purified by denatured nickel-resin method using 6M urea
> (expressed protein in inclusion bodies).
> 
> There were tons of protein expressed in the first two elution fractions
> (2ml per fraction), as a single band (around 35 kd) by SDS-PAGE.
> But, after I did the dialysis against refolding buffer (Tris.HCl 50mM,
> NaCl 50mM, CaCl 10mM, pH 8.0) and filter through a 0.22 um disk filter,
> the protein was gone (as shown by BioRad protein assay and ELISA).
> 
> I am wondering what had happened to those protein. I noticed some
> precipitation in the dialysed solution. As to the refolding buffer,
> I got the recipe from a paper whose authors had successfully used it to
> refold their denatured protein.
> 
> Thanks in advance if someone could lend some help.
> 
> Alex Chang
> Pathology
> University of British Columbia
> achang at hivnet.ubc.ca

Alex, you`ve got particulary difficult task.
I belive, that antibody re-folding would grately depend on how properly
S-S briges are formed during dialysis. From what you described, it seems that all your
protein forms misfolded precipitate.
It was reported that basic conditions could help during refolding. High protein
concentration usually increase portion of misfolded aggregates.
Therefore, I`d start from 50 mM Tris pH 10.5, 150-200 mM NaCl, 1 M urea.
I`d try to avoid addition of EDTA, and DTT or BME, as well as redused/oxidized
glutathion. Lets pH of dialysis buffer and presence of BME in elution buffer
do the job. I`d also reduce protein concentration or add more up to 0.5 M NaCl,
alternatively you could bring 10-20% glycerol in your solutions.
The next step I`d dialize against 50 mM Tris pH 8.0, 300 mM NaCl. If the protein
would survive in the solution the first step, then after the second step you could be
100% sure you`ve got whaty you need.
In all cases dyalisis should be carried out w/o vortexing,
mixing, and about 30 hr. with refreshing the buffers.

Hope it helps. 
Nikolai
http://128.252.119.253






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