His-Tag purification - contaminated protein

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Elayoe
Elayoe's picture
His-Tag purification - contaminated protein

Hallo,

I have a problem with the purification of my His-tagged protein:
After the purification I can see additional bands in the gel, so I don't have 100% pure protein. I used to have no problems with that. I learned the purification not where I do it now, but I use the same buffers and do everything exactly the same way.

The additional bands I see are:
1. bands smaller than my protein
2. bands smaller than my protein that I can already see in the lysate
3. bands bigger than my protein I can see in the lysate
4. bands bigger than my protein which are at the very top of the gel (therefore must be very hugh)

1. could be due to degradation but it never happend before... All buffers are cooled in ice before usage so the room temperature shouldn't make a difference.
2. and 3. could be because some interaction or unspecific binding, but why didn't that happen before???
4. could be DNA/RNA????

I need to have pure protein for my assays so I need to remove all these additional bands!

A bit more information about how I do the purification:
I express my protein in E.coli DE3 BL-21 RPL and lyse them with the following buffer:
20 mM Tris; 500 mM NaCL; 10% Glycerol; 20 mM Imidazole; pH 8.0.
And I am also adding 1 mM PMSF, 2 mM mercaptoethanol, 1% Triton, Lysozyme and DNase.
I keep it stirring for 2 h at 4C and after centrifugation load it onto a Ni-NTA-column and wash with cold buffer before I elute it with cold buffer.

As I said, I do the protocol exactly like I learned it but now I get these additional bands and have no idea what I can do...

Hope you can help me. If you need to know anything else, just ask...
Thanks in advance.

kriodos
kriodos's picture
I supposed that you see the

I supposed that you see the bands in a SDS gel electhrophoresis so I think the bigger bands can no aggregatinos from your protein but can be glycosilated forms. wich kind of protein is? membrane? cytosolic? First at alll I were did a western blot of the sample with anti-his antiboy for see if the band have the his tag or not. Second, triton X-100 1% is too much and can solubilize too much non desireed proteins. try with 0.1%. After sample aplication wash wih 4 volumen of 50-100 mM imidazole. and after a gradient up to 500 mM.
good luck

rksreeji
rksreeji's picture
hello,

hello,
the problem I am facing is that the protein expressed with the his tag fusion in not binding at all to the Ni NTA beads. can anyone suggest why this happens? Its new beads and the protein is expressing properly and has detected the his tag using the anti his tag antibody.

kjosephs
kjosephs's picture
In response to the first part

In response to the first part of your post, some times changes in the level of contaminants is actually a result of reduced expression levels. Can you verify that your expression is as good as it was in the past? Did you change anything in the expression, like using colonies from an old transformation or glycerol stock instead of a fresh transformation?

If you can establish that your expression is as robust as before then you can focus on the Ni purification. Before you invest tons of time on this, it is worth boiling some of the Ni-resin that your protein appeared not to bind in SDS-page buffer and running that on a gel. This is a quick check to make sure that the protein really didn't bind and that it isn't failing to elute because it aggregated.

If you still suspect the Ni capture step, you can try checking the pH of the lysis buffer. Ni capture depends on deprotonating histidines and so the binding is not efficient if the pH of the buffer is below 7.5 and is better at 8.0. If the composition of the lysis buffer seems ok, I would suggest testing the Ni-resin and buffers with any purified Ni-tagged protein if you have it. You can even spike it into your lysate.

rksreeji
rksreeji's picture
hello,

hello,
thanx for the suggestions, I have taken care that the pH of the buffer is 8.0 through out my purification and why I am sure that the protein is not binding is that I see most of the protein in my flow through. the binding is just 5% inspite of the fact that I take much more than required slurry for the purification. All steps are done at 8 M urea but still there is hardly any improvement. What do you feel about this?

kjosephs
kjosephs's picture
OK. It's either the protein,

OK. It's either the protein, something in the buffer, or the resin. I have to believe that if you are using new resin for each experiment that it is not the resin. Besides pH the other buffer components that effect ni capture are reducing agents and imidizole. The concentrations that you described are typically tolerated, so I'm suspecting the protein.

Since you have done the 8M Urea experiment, it's not an aggregation issue or problems with display of the His tag. I think you should verify that the His tag exists by western blot of the flow through using an anti-hexa his primary antibody.

I guess its possible that you are getting proteolysis of the His tag during the lysis, but my hunch is that this will trace back to an expression problem (you may actually be purifying a host protein of similar molecular weight-something that is easier to do when you start using more and more Ni-NTA agarose). Either way the western blot will be an informative experiment.

Let us know how it goes, and good luck!

rksreeji
rksreeji's picture
Hi,

Hi,
I have done the western blot using anti his tag antibody. it gives the correct band size after detection. also I have sequenced the DNA (N terminal end of the clone). It indicates the presence of 6 His residues (His tag). And my expression vector carries His tag at both N terminus and C terminus. But still the binding dosent occur even under denaturing condition. That makes it all the problematic, I am planning to change the vector system and try GST tag.

R Bishop
R Bishop's picture
rksreeji,

rksreeji,

I really think you need to find a labmate or someone in the area that has a HIS-Tagged protein and run it through your procedure as a positive control. If its the resin or a buffer you are going to spend days re-cloning etc.

For example Abcam has a control E. coli lysate available

http://www.abcam.com/E-coli-Positive-Control-Whole-Cell-Lysate-expressing-6X-His-tag-protein-ab2431.html

Rb

kjosephs
kjosephs's picture
I have to say I agree with Rb

I have to say I agree with Rb. If you had this working as a His-tagged construct that gave you what you wanted in a single step, it would be a shame and a lot of work to have to change to GST. While GST capture/elution is more specific, the binding is much slower and not as complete as His-tags and you will always get host GST in your preps so to get >95% purity will require additional steps. Not to mention the time for the re-cloning and protease cleavage if you can not use the GST fusion in your experiments.

What Ni-resin are you using and have other people in the lab used the same exact resin lot and bottle successfully?

rksreeji
rksreeji's picture
hi thanx for your valuable

hi thanx for your valuable suggestions, I will definitely try to do it with a control his tag protein to make sure that conditions are fine or not. Thank you
rksreeji

R Bishop
R Bishop's picture
Any luck fixing your problem?

Any luck fixing your problem?