svetlov at oncology.wisc.edu
Thu Jan 29 11:27:56 EST 1998
In article <01bd2c0c$e8880560$2d187889 at henri>, "Henri M.H. Spronk"
<henri.spronk at bioch.unimaas.nl> wrote:
> After expression (in E.coli) of a recombinant human protein as part of a
> 6His-DHFR-fusion protein and purification using a Ni-NTA-column, the
> protein is insolluble. The protein is soluble in a solution of pH 12, but
> not at 7.5. Does anybody have experience with protein solubillization?
If your elution protocol was based on lowering pH, try using imidazole or
EDTA instead. As for solubilization of the precipitate you've got already
you can refold your protein after solubilization in 8M urea or 6 M
guanidinium chloride/isothiocyanate by dyalysis in presence of low
concentrations of sarcosyl or paramolar concentrations of Arg (other aa
such as Trp were also used in refolding of proteins prone to aggregation
upon dremoval of chaotropic agents). Try using some intermediate pH for
that (between your 7.5 and 12), if you are lucky you might get your
refolded protein stable at smth. like 9.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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