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-   -   His-tagged protein purification from yeast (k lactis) supernatant (http://www.molecularstation.com/forum/protein-forum/26992-his-tagged-protein-purification-yeast-k-lactis-supernatant.html)

Tiwari 01-03-2008 05:44 AM

His-tagged protein purification from yeast (k lactis) supernatant
 
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.

Thanks!

DK 01-03-2008 10:47 PM

His-tagged protein purification from yeast (k lactis) supernatant
 
In article <[Only registered and activated users can see links. Click Here To Register...]>, Tiwari <[Only registered and activated users can see links. Click Here To Register...]> wrote:

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




Tiwari 01-04-2008 01:08 AM

His-tagged protein purification from yeast (k lactis) supernatant
 
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, [Only registered and activated users can see links. Click Here To Register...] (DK) wrote:


DK 01-04-2008 08:03 PM

His-tagged protein purification from yeast (k lactis) supernatant
 
In article <[Only registered and activated users can see links. Click Here To Register...]>, Tiwari <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Well, that means you need to get rid of the medium and all of my
suggestions are potentially applicable.

DK



Dr Engelbert Buxbaum 01-16-2008 06:48 PM

His-tagged protein purification from yeast (k lactis) supernatant
 
Am 04.01.2008, 16:03 Uhr, schrieb DK <[Only registered and activated users can see links. Click Here To Register...]>:



Yes, I think so too. I'd probably try to concentrate my extract in an
Amicon pressure cell, add a suitable buffer and repeat a couple of times.
That should remove most of the low MW contaminants.

Even better of course to pellet the cells in a centrifuge, resuspend and
then lyse in a defined medium. I never liked the idea of proprietary
reagents where you don't know what the hell you are getting into.

GormEnOrm 01-18-2008 12:14 PM

His-tagged protein purification from yeast (k lactis) supernatant
 
On 3 Jan., 06:44, Tiwari <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Hi Tiwari,
I have had the same problem with Baculovirus System. Media contains
both a chelating agent and a imidazole-like molecule (probable
histidine in high amounts) :-(. For a long time we have dialysed
samples for three days against 5 x 8 L buffer. Such a waste of water,
time and chemicals. But there are smart solutions.
You could try ion-exchange column to concentrate protein and get rid
of oppositely charged contaminants. Then the sample volume would be
lowered to 10 - 20 mL, which can be dialysed or desalted otherwise. If
you purify an enzyme and have an assay for detection, this can quickly
be optimized.
The best solution is a CrossFlow machine. We just bought one in my
lab. It takes in fx. 1-600 mL sample (we only have 600 mL as max, but
machine can take more), concentrates to 32 mL and diafiltrates (10 kDa
cut-off) with 160 mL buffer. This results in approx. 45 mL
concentrated sample with a 95 % recovery of protein and a 95-98 %
reduction in small contaminants, just ready to go to the Ni-NTA
column. All done in 3 - 4 hrs. Nothing can beat that.

- Regards

Gormicon

incrediblyhigh@hotmail.com 02-20-2008 02:39 PM

His-tagged protein purification from yeast (k lactis) supernatant
 
On Jan 4, 1:08*am, Tiwari <[Only registered and activated users can see links. Click Here To Register...]> wrote:

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


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