Recovering possible soluble protein from inclusion bodies.

[angel_demon12]

Hi all,

I am working with a horrible protein that is expressed in E.coli in the form of inclusion bodies Protein refolding has been problematic and I want to see if there is any soluble protein within the inclusion bodies.

My PhD supervisor has told me that sometimes there is some protein within the inclusion bodies that has native-like secondary structure and that this can be recovered using a low concentration of urea.

I have tried to find an article describing this but instead of finding what I want, I get bombarded with articles on protein refolding.

Does anyone have experience with this, or does anyone know a good article that I could look at.

Thanks for your help!

[relaxin]

The majority of the expressed protein is in the inclusion bodies. That is why most people will go for refolding than trying to purify the small amount of "native" protein. There are many posts in this forum dealing with protein refolding. You should be able to refold your protein.

[angel_demon12]

Thanks, but as I said: protein refolding has been problematic.

I have spent a VERY long time trying to refold this protein and would like to explore another avenue.

My protein is very difficult to refold (due to the lack of glycosylation, the 5 disulfide bonds and the fact that it is a dimer) and all expression systems so far tried have resulted in inclusion bodies. A yeast expression system is currently being prepared but is taking a long time. While I am waiting for this, I would like to try recovering protein from the inclusion bodies.

So all I am after is some sort of protocol describing how best to recover the small percentage of protein that is soluble/correctly folded within the inclusion bodies.

[kayvee]

Firstly, there are several ways to wean a protein into soluble fraction. I am guessing you went through most of them like temperature, IPTG concentrations, etc. A few that you might have overlooked include:
- using different kind of cell strains
- clipping or adding amino acids on either ends of the protein
- using a different vector - one with a promoter different from what you are using
- using a different tag - if you can have a tag to start with

Another thing that you can do with the protein that you already have is this: use a small percentage of detergent to get it out of the inclusion bodies. Triton is a class of detergents you can start with. Search for terms like triton, df-16,etc in the forum and you should get some head start.

Good luck.

[Compton]

Hi all,

I am working with a horrible protein that is expressed in E.coli in the form of inclusion bodies Protein refolding has been problematic and I want to see if there is any soluble protein within the inclusion bodies.

My PhD supervisor has told me that sometimes there is some protein within the inclusion bodies that has native-like secondary structure and that this can be recovered using a low concentration of urea.

I have tried to find an article describing this but instead of finding what I want, I get bombarded with articles on protein refolding.

Does anyone have experience with this, or does anyone know a good article that I could look at.

Thanks for your help!

Can you briefly describe how you have tried to refold the protein? During the last 6 months I've expressed 12 proteins in E.coli using the SUMO expression system from Invitrogen. My standard protocol has been:

1. Isolate the inclusion bodies
2. Solubilize in 8 M urea or 6 M GuHCl (2 protein required GuHCl)
3. Purify with Ni-NTA resin under denaturing conditions
4. Refold against 3 M urea, 20 mM tris, 1 mM DTT, 150 mM NaCl, pH 8.0 using a normal dialysis chamber
(5. remove the SUMO tag with SUMO protease)

6 of the proteins were able to refold at 2 M urea but 6 required 3 M. Although I have to admit that I don't know (or care) whether the actual protein of interest folded or not - the SUMO protein folded and that enabled me to cut it off from my protein of interest. I'm only interested in the denatured form of the protein for ELISA studies. Anyway, if SUMO can refold in 3 M urea, so should other robust proteins do as well.

[bform]

Sarkosyl works well to solubilize inclusion bodies. You may give that a try. here is a reference - J Cell Sci. 1993 Jun;105 ( Pt 2):481-8. PMID 8489015

I've tried sarkosyl and it has worked to purify and/or increase yield of protein that has gone into inclusion bodies. For one protein though, the sarkosyl purification seemed to work and I was able to purify protein, but the protein did not appear to be active (although I am not sure of the reason).

What others mentioned may work as well, I'm no expert. I will be trying to cleave N and/or C terminal amino acids to see if that helps on my problem protein.

[angel_demon12]

Ok, so things I have already tried include:

Different constructs (aa mutants, constructs with different N- and C-terminal tags).
Different vectors.
Different E.coli host strains.

All resulted in inclusion bodies (even the construct with an N-terminal signal peptide that directed it to the periplasm).

Refolding has been carried out and the most important steps are described below:

1. Solubilisation of inclusion bodies with 6M GdmCl.
2. IMAC purification.
3. Reduction of cysteines with DTT.
4. Gel filtration purification - at this point the protein is approx. 99% pure).
5. Refolding by rapid dilution (although other methods have also been tried).
6. SDS-PAGE, Native-PAGE and activity assays to monitor refolding.

Unfortunately, even after extensive refolding optimisation, very low yeilds could be obtained (less than 5%).

Everything and I mean EVERYTHING was attempted in order to purify the active protein from the misfolded protein, however, it was not possible to do so.

I have almost 2 years experience in protein refolding and I can tell you that this protein just does not want to refold. The refolding problems are associated with the fact that the native protein is:

A dimer with a monomeric size of 60 kDa (dimer is 120 kDa).
Each monomer contains domains.
Each monomer contains 5 disulfide bonds.
The protein is post-translationally modified - glycosylation at 4 sites and the addition of a GPI anchor at its C-termini.

I do not wish to continue with refolding attempts and wish only to try and recover any protein with native-like secondary structure trapped within the inclusion bodies. I do not want to solubilise the inclusion bodies themselves.

I hope this has made the situation a little clearer.

Thank you for your ideas. I will now consider the possibility of using sarkosyl or triton-x. If you have any further ideas please let me know.

[Georgia upenn]

I am also working with protein expression for long time now. Lately I had to express 16 mutant proteins and all of them came to the insoluble fraction. I am using the pQE30 vector from Qiagen so the yield is high.
I had a lot of trouble to refold these proteins especially because they have 16 disulfide bonds and size of 28KDa.
Finally I tried the Refold-Kit from Novagen. Basically it is an ELISA plate that has 96 different buffers for refolding. It costs close to 400$ (If I rememebr correctly) but it really saved me. I found two different combinations of buffers that refolded my protein.
To check the refolding of your protein can you do CD? Is the crystall structure known? Another way is to run an anion or kation exchange column. If you have an heterogenoeus population of protein (folded and missfolded) then you will have multiple picks. If yout protein is 100% folded you will have a single sharp pick.

[angel_demon12]

I check refolding using an activity assay. I do not have access to a CD instrument and the crystal structure is unknown. I have tried all methods of purifying the correctly folded protein from the misfolded (all chromatographic methods, precipitation methods, prep-native-PAGE etc.) ion exchange attempts did not work (using all columns available and a wide range of pHs). Gel filtration revealed multiple species: monomer, dimer, and multiple aggregation states. Unfortunately gel filtration did not give satisfactory separation between the monomer and dimer.
In response to your idea about the Refold-kit, I have made similar 96-well plate screens and use this to optimise my refolding buffers.

[angel_demon12]

Wow! - 16 disulfides (so 32 cysteines involved) in a 28 kDa protein! And I thought I was having a hard time getting correct disulfide pairings for 5 disulfides. Did you check the disulfides eg. by MSMS, ABD-F staining etc.?

I just find it hard to believe that you could correctly refold such a protein.

[Georgia upenn]

To chack the refolding of my protein I am using different tecnhiques, like:

1. reactivity with monoclonal antibodies,
2. CD
3. Anio exchange chromatography and affinity chromatography (heparin binding)
4. Functional assay comparing with Pichia expressed protein
5. MALDI using mercury. If there is a free cysteine then the mercury reacts with it and I have increase of almost 200mass units.
It is tough, but the combination of all these can give you some answears....

[kayvee]

With those many cysteines and disulfide bonds present in your protein, are you using one of the origami-based E. coli strains from Novagen? I had to purify a 13kDa protein that has 16 cysteines and was able to do it only when I use Rosettagami cells.

[ANLA]

bform wrote that: "Sarkosyl works well to solubilize inclusion bodies. You may give that a try. here is a reference - J Cell Sci. 1993 Jun;105 ( Pt 2):481-8. PMID 8489015"

This would be interesting to read more about, but I cannot find anything about sarcosyl in that reference by Frangioni & Neel. However, in Analytical Biochemistry (1993) 210: 179-187, the same authors describe the solubilization of GST-fusions with sarcosyl.

[kayvee]

In fact, for GST-tagged proteins, sarkosyl is mentioned at the end of the GST handbook from Amersham.

[relaxin]

A recent publication describes the expression of foreign protein in soluble form by fusion to E. coli thermostable protein (Trigger Factor) through a ubiquitin bridge. The foreign protein can be excised with a deubiquitylating enzyme, Usp2-cc.

For details, please read:

http://www.ncbi.nlm.nih.gov/pubmed/1847 ... d_RVDocSum