Protein refolding help

[dhawker]

I don't really have biochemistry experience in this so I was hoping someone could give me suggestions. I have isolated an enzyme from whole animal tissue, but it lacks all activity. I ran a couple of native gels and it looks like it is denatured (bands appear the same as those boiled in sds buffer prior to loading). I have a paper where they refold the protein using urea, but they are refolding inclusions bodies from e. coli overexpression (long story why we use tissue...). First question, does refolding even work for proteins isolated from whole tissue? Almost all of the papers I have found are always working with proteins generated in ecoli. If it's still doable, I'll ask the rest of my questions...

 

What is a good concentration to have my protein at before starting? Low? How low?

 

In the protocol I have, they elute from their final column under denaturing conditions (8M urea), then dialyze away the urea step-wise (4, 2, 0 M). Would it be possible to just dialyze straight into 8 M urea, then follow their protocol?

 

What would you recommend for dialysis protocol (sample volume v. buffer volume, dialysis duration, number of buffer exchanges per concentration, etc...)

 

At this point I have a ton of pure protein... just denatured, so I have room to mess around with it I guess. Hopefully I'm not asking too much, but I would appreciate any and all suggestions.

 

Thanks!

[CharonY]

Once denatured it is often not easy or possible to have them refolded properly again. To be honest the best guess would be try to reisolate it under non-denaturing conditions.

[dhawker]

You see, that's the problem...they were never isolated under denaturing conditions in the first place (at least intentionally). The purification protocol is extremely long and taxing and so if it is possible to renature I really want to give it a try. As far as I can tell the enzyme is intact, just inactive and denature (SDS-PAGE looks perfect). I know the yield may not be great, but it would be better than going through the entire protocol again. Supposedly it is possible to refold this exact enzyme, so assuming this publication is legitimate (which is still in question in my opinion since they won't return my emails) can anyone give suggestions???

[CharonY]

Edit: I was rambling a bit.

 

To clarify: if you did not denature your protein during isolation a denaturation in urea with subsequent renaturation does not make much sense. The question is whether the proteins are really denatured. A native gel gives only limited hints, depending on the protocols used. The question would be how much of a difference you would expect from a native protein compared to a SDS denatured. Confirmation state are harder to estimate, but you could check the expected charge, under the given conditions (unless your native protocol introduces a charge externally, of course). The lack of activity is for me a more compelling argument. However, I assume the protocol used is known to work and not to affect the activity of the protein?

Also if a MS is available you may want to verify that your protein is really the correct one.

 

If it is really denatured you should try to track back and find out what it was and try to remove that, rather than denaturing it with urea and removing it again does not sound too promising. There are several ways to proceed, the easiest is simple removal of denaturing agent and rebuffering. One thing is to determine the right conditions in which it should refold (often it is a Tris buffer with various oxidizing or reducing additives). Depending on the properties of the denaturant you could simply use spin columns to remove the original buffer and replace with renaturation buffer. If the whole isolation process somehow caused the denaturation non-chemically it is going to be very tricky, as these may have changed the properties of the protein irreversibly.

Edited June 12, 2010 by CharonY

[Greippi]

CharonY pretty much covered it.

 

I might be able to come up with another possibility if I knew what the enzyme was, and how you're measuring for activity.