His tagged protein folding - Confused -Please help

[KLIK]

Hello

Iam working on recombinant fusion protein which has HSP70 as a part of it. (This protein has His-tag too). protein was expressed in bacterial cells and purified using Nickel column under denaturing conditions (8M urea). In order to refold the protein after purification elute fraction (protein eluted with 8M urea pH 4.0) was dialyzed against 10mM Tris (pH 8.0), 0.1% Triton X-100 followed by dialysis in PBS (pH 7.4). Does this procedure refold the protein or not? I need some help in this matter. can anybody answer my question. i appreciate your help very much.

[RandomFactor]

Dude,

 

The folding of the protein is highly dependant upon the nature of the amino acids used in the protein, I don't believe with the information given it would be possible for anyone to tell you whether that would refold...

 

If you have access to an NMR machine, perhaps you can try a variety of conditions and use a 2D HSQC experiment to check if the protein looks right? You would have to make more protein which was 15-N labelled in order to do this, and would aslo have to do an HSQC on a 15N labelled "control" sample, meaning without the His Tag. These can then be compared and if the spectra near enough overlap...then "Happy Days" .

 

Hope this helped.

[KLIK]

Thanks for your reply.

I dont have access to NMR (well our department is not well equipped).i dont have much experience with protein chemistry so I read in some of the protein purification protocols books - it was said that when protein is purified under denaturing conditions with urea, removal of urea by dialysis will bring the protein to native state. so using the procedure that i mentioned before i was trying to do that. My experiment is to use the pure protein for immunizing mice and look at their immune response. Anyway i will try to get some help NMR from someone. Thanks again for your suggestions.

[RandomFactor]

Yah, mostly if you dialyse with progressivly less concentrated urea, you will return back to the native protein, but the problem here is that, the chaperones acutally help with the folding...

 

I'm not sure what you mean by "HSP70 as a part of it", but if you mean that they are functioning as they should, then maybe the technique you described would work.

 

The problem is that you don't have any proof of it, so...the only way I can think of to get proof would be to use an NMR machine...unless you want to make X-Ray crystals, and work out the 3D structure...but lol it's better not to go there, because that would take ages. The NMR route would take you the time needed to make the samples and then probably a few hours on a basic NMR machine, and a little bit of time to compare the spectra...so it really is (in my opinion) the most time efficient way of getting direct proof that the protein has folded up properly.

[KLIK]

when i said "HSP70 as a part of it" i mean to say that my gene X ( produce protein X when expressed) of interest was fused to HSP70 and the final protein obtained was recombinant fusion protein X-HSP70.

[Yggdrasil]

It is not always possible to refold a protein when it is purified under denaturing conditions. If you don't have access to an NMR machine, you could try obtaining a CD (circular dichroism) spectrum since that can provide a good enough assessment of whether your protein is correctly folded.

 

If dialyzying the urea out of the eluant doesn't correctly fold your protein, you could try refolding the protien on the Ni column. After binding the protein to the column, wash with progressively lower concentrations of urea. Then was with buffer containing no urea and elute in a buffer containing no urea.

[KLIK]

hey

Thanks for your suggestion. Can you please tell me little bit more about CD (circular dichroism) spectrum. Iam not at all familiar with this.

[RandomFactor]

Circular Dichroism analysis of a sample is a spectroscopic technique whereby you use circularly polarised light, and then shine it through your sample, and then it gives an ellipically polarised beam at the other end... (very basic description).

 

By analysing the ellipticity of the light at the end...you can get an idea of the helical character of the protein, or B-sheet character...

 

It is not as direct as the NMR method, and if you were writing a paper, I don't know how it would be taken as proof of correct folding...but its something which you can use in the lab as a good indicator...it's a much faster way than NMR...

 

I think I will wait for Yggdrasil to explain it more fully, before adding more (slight bit busy at the moment).

[KLIK]

Thanks for your explanation