[Protein-analysis] Re: His-tagged protein purification from yeast (k lactis) supernatant

incrediblyhigh from hotmail.com via proteins%40net.bio.net (by incrediblyhigh from hotmail.com)
Wed Feb 20 09:39:37 EST 2008


On Jan 4, 1:08 am, Tiwari <kstiw... from gmail.com> wrote:
> The media contains no EDTA/EGTA/DDT/BME nothing just yeast extract +
> peptones + galactose. Media alone was able to chelate Ni from resin.
>
> On Jan 3, 5:47 pm, d... from no.email.thankstospam.net (DK) wrote:
>
> > In article <6ecae0f2-19ab-4360-8ec6-0b1fa43df... from x69g2000hsx.googlegroups.com>, Tiwari <kstiw... from gmail.com> wrote:
>
> > >Hi Guys,
>
> > >I am expressing the recombinant protein 6xHis tagged at C' terminal
> > >secreting in YP-Galactose media by K lactis. My problem is whenever i
> > >try to purify by using Ni-NTA column, it turns the column white as if
> > >supernatant is chelating Ni from the column. I tried to condition
> > >supernatant by adjusting pH 8.0 and Salt upto 1M with no success.
> > >Please suggest me what should i do. Ammonium sulphate precipitation
> > >result in inactivation of protein. i cant do dialysis economically to
> > >huge culture volume.  so these will not be a good option for
> > >conditioning media.
>
> > Try adding 10 mM Mg2+. If you are lucky , the thing that strips Ni2+
> > also binds Mg2+.
>
> > Besides AS precipitation, another option to concentrate proteins
> > and get rid of the media components that strip nickel is
> > hydroxyapatite absorption. If "your" protein binds to an ion exchanger
> > under medium's salt condition, that's also a good high capacity
> > option (even if it does not, diluting medium with water before ion
> > exchanger absorption is yet another option).
>
> > DK
>
> > >Thanks!

Have you tried talon beads? They might perform better... What about
changing the tag to say, a strep tag?


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