his tag protein purification

Hi, my name is Humberto. I'm am currently working with the TOPO TA cloning kit, which I used to obtain my recombinant protein. Using the vector pBAD TOPO, the recominant protein I get is of about 13 kD taking into account the V5 epitope and tandem section of histidines. I performed all the pilot _expression essays to determine the time and concentration of arabinose to best induce the production of my protein from this vector. Once I did this, I grew a 50 ml culture to use in the purification of my protein using the probond system procedure.

Before I started to purifie it, i resuspended the pellet of cells in native binding buffer from the probond system( because I want to preserve the activity of the protein) sonicated the cells and follow all the native purification steps...so I obtained the supernatant and performed a SDS-PAGE and used the comassie blue staining to visualize the protein. I was able to see the protein, loading 10 ul from the 8 ml volume of the supernatant. The intensity of the band was high. Then I prepare the column with the probond resin and once ready I loaded the lysate just like the protocols mentions, and did all the steps. In the native binding buffer I didnt use imidazole. just in the wash and elution buffers. I collected the wash supernatants and the 1 ml elution fractions and did another sds page. I loaded 12 fractions and a sample of the washes collected. The results I got were that I could not see the protein in the elution fractions except in the elution fraction number 1 in which I barely see it with a bit of imagination, but not in the other 11. I also see a few high molecular proteins in the first 3 elution fractions. But I think that my protein is not binding the resin because in the first supernatant which is the one that I obtain after the binding step, I see my protein with the same intensity as the first sds page I ran. And in the other washes i see the protein too, with less intensity though.

I checked the troubleshooting section of the probond manual, but one of the answers I can come up with is that the folding of my protein may influence the proper binding with the histidines.

I checked the solutions. pH and everything. Do you think that the concentration of my protein in the lysate could be a reason or the concentration does not influence in any way the binding?

I dont want to try the denaturing procedure of the probond system because I want to preserve the activity of the protein. Does any one have any suggestions to solve this problem?

I'd really appreciate it.

thanks in advance,

Humberto O.

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Historians believe that in newspost
<[Only registered users see links. ]> on Wed, 22 Oct 2003,
[Only registered users see links. ] penned the following literary masterpiece:

If the His's are folded within your protein then they are not available
for the column to actually 'see' to bind to.

Assuming the above isn't a problem then what happens if you dilute your
sample say 5 fold and try loading that?

It may simply be that the initial protein 'soup' is really too high a
concentration, plus the presence of nucleic acids in it, and is stopping
binding.

Duncan

--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

I thought the premise was that 'masking' was effected, in this case, by
folding of the protein within itself and not related to the concentration of
molecules sorrounding it. Well....

--
Rolands G. Aravindan
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Duncan Clark wrote in message <6xMlqADaK7l$EAoQ@[127.0.0.1]>...