If you cant find condition for soluble expression of your protein i would suggest that you switch to a His-tag and use IMAC purification. The reason is that you can (1) use inclusion body purification protocols which already yields a high purity and (2) IMAC is compatible with 8 M urea and you can refold your protein on the column. This has several advantages compared to refolding in solution. Nevertheless you would probably have to investigate different conditions and additives.

I think 8M urea might have denatured GST so it lost its ability to bind. Try milder concentration of urea coupled with sonication.

If the protein is in IBs then it is unfolded. I would use an alternative tag such as a HIS tag for making protein, so that if it forms IBs then ,you can purify it after using 8M urea and then refold it. Alternatively, you can try to refold e GST- tagged protein from urea first, and then see if it will stick to the Resin.

What do you suggest for refold the protein? Thanks

The biggest concentration of urea that can admit GST for working as fusion-tag is approx. 1.5 M, higher concentrations denature the protein and it is not possible to use this system for purification any more. If your proteins are very unsoluble and you need chaotropic agents for performing the purification, the most practical action is to use a different fusion-tag, for example his-tag.

Dear Claudia

The concentration that you used is conventionaly used for protein denaturation in 2DE sample preparation. If GST is denatured, than it can not worked properly.

Renaturation is poosible but you must drain out urea completely.

The best way is starting the process from the first by lower urea concentration that mentioned by Ana at the previous comments.

Optimum refolding conditions vary from protein to protein. In my experience dialyzing the urea out in small increments ( start with 7 M urea, dialyze overnight in 2-L buffer then switch to 6M, 5M, 4M, ...1M, 0.5 M and finally into a buffer with no urea) usually works well. If your protein needs co-factors for proper binding make sure you add them to your buffers. If you still have trouble folding there are several companies that sell refolding kits for you to optimize the best conditions.

Have you ever optimized you expression conditions, like IPTG concentration, induction temperature, and incubation time? I have some proteins that go into IB at 37 degree, but soluble at 16 degree. If you have to purify from IB, try above suggestion to change the Tag. I succeed with his-tag. The refold condition is the most tricky part, it depends on your protein, how big is your protein, how many disulfide bonds your protein has etc. the robust method I tried is rapid dilution with cystine/cysteamine. Other method include on column refold, step dialysis, etc with GSH/GSSG or other strong redox pair agents. U will figure out which method suits you.

There is a company that might be able to help you to refold your proteins. The website is http://www.mefold.com/index_en.html. Good luck!

I agree with Marcia and Ana that you will need to refold the protein to get binding to GST, and that a His-tag would be better (as it does not require the protein to be folded). You could always use a His-GST vector (e.g. pET41a, pGAT3). Before refolding, you can get really good purification by washing the inclusion bodies with lysis buffer (preferably including 1% (v/v) Triton X-100) and re-sonicating: this helps to remove other proteins and unbroken cells that will contaminate your prep.

The refolding itself is something of a black art, and the correct approach varies from protein to protein. There are very good reviews available that go through common approaches. Alas, for some proteins, nothing really works except for expression in a suitable system. If you do keep the GST-tag, don't be confident that you have refolded protein until you have removed the tag: GST tags are great at keeping misfolded mush in solution.

Good luck!

Maybe detergent-based extraction could help you. I have used CHAPS and I recovered most of my protein from inclusion bodies in E. coli, in native state. Such protein was His-tagged and purified by Ni-NTA resin. After elution and wash it retained its ability to bind nucleic acids. Other useful detergents could be Nonidet, Igepal, Tween, but I have not tried those and in each case you may have to standarize their concentration for extraction. Hope you find this helpful.

Keep in mind that GST uses enzyme-substrate based purificatio. Althought. 8Mure is reccomendable for proteins that are insoluble or in inclusion bodie, it is totally useless as you would have denature the enzyme (GST)therefoore it would not be able to bind to you column. It. Wouldbe better to get your recombinant be expressed. As a soluble protein, if fused to GST. This however does not apply to His-tagged recombinants, I would actualy recommend 8M Urea (and other additives of course, request if you want my buffer). If it is a his tagged fusiojn refolding is possible on column ( request request if interested) whilt GST fused will not be Able on column, unless using dialysis

My understanding is that the GST purification doesn't work with denatured proteins. However, there are several options for trying to solve the problem. One option would be to try to refold the fusion protein before binding it to the GST affinity resin. You can find different protocols for doing this and there are even commercial kits available for protein refolding. I have refolded protein in the lab simply by dialyzing out the urea in cold buffer. It is possible that the refolding won't work well for the fusion protein. So a second thing you can try, is to try to get the fusion protein to express as soluble by lowering the temperature during expression. Also, you could try different lysis buffer conditions that promote protein solubility including use of detergent. A third approach requires changing to vector to express a His tag fusion protein. These proteins can be affinity purified while denatured. I saw a publication that addresses the problems of this question in Cold Spring Harbor Protocols. (Einarson M.B. 2005). http://cshprotocols.cshlp.org/content/2007/4/pdb.prot4738.full.

In my experience, I have used ion exchange chromatography to purify proteins that express as inclusion bodies. My advice is to make sure that the lysis by sonication is done well enough, as most proteins will solubilize resulting in a large amount of purification. This may not be as important with affinity purification, however, and Einarson indicates that too much sonication can have negative effects on GST affinity binding, though I don't know if this is a factor if the protein is going to be refolded. When purified inclusion bodies in the lab, I sonicated 10 second pulses 10 times, with one minute of cooling on Ice between pulses. I centrifuged to collect the inclusion body pellet, resuspeded the inclusion bodies and repeated the sonication procedure and centrifugation. This procedure led to a surprising 95% purification of the expressed protein before doing any chromatography.

For refolding, there are several kits one can buy that have several different buffer systems already prepared for you to try. You can either try to slowly reduce the concentration of urea with the prepared buffer systems in the kit, do dialysis, or dilute directly into the buffer without slowly changing the urea concentration. I would try the decrease in urea technique slowly first and direct dilution to less than 0.6M Urea as others have described. If your protein will stick to an ion exchange column, then you may be able to refold it on a column. But as I and others have said before, you are better off win a HIS tag. But since you have already made the construct, refolding, and then seeing what sticks to your affinity column for subsequent purification is the most timely and easy thing you can do.

thank you everybody for all your answers. Im very new in this are. This is the first time I wok with proteins, but all your comments are very useful. since I already have my fusion proteins (8 of them) with the GST tagI will try to dilute out the urea, or use one of the protein refolding kit that I found...I forgot to mention that before I passed my sample through the column I diluted it 8 times in PBS+tritonx100. What about if I used 4M urea instead of 8M and then start to dilute it out?

Hi Claudia,

I think using less concentration of urea is indeed good idea , u can also make concentration gradient ( like 3,4,5...) and see which work best for you. good luck !

The 8 M urea is needed to dissolve everything and get efficient extraction of your protein. The 4M urea and the 8 M urea in terms of denaturation are the same. So people use 8M urea. There is no real advantage to using 4M.

In terms of forming inclusion bodies, have you changed the expression conditions to see if you can get soluble protein? Ie like temperature, time of induction, etc. each construct is unique and the conditions need to be empirically determined.

The point in using concentration gradient of urea is in terms of solubility of the protein, if 4M of urea if sufficient to dissolve GST from other cellular proteins, we don't need to use higher concentrations of urea.

That is true and a good point Mohammed. Then one might want to extract first with 4M followed by 8M to see if any additional protein comes out.

Thanks Marcia, hope this will help Claudia!

Hello Claudia,

I would like to say that I used Tween 20 and it worked, so you may try it.

Good luck

Try to obtain your protein in soluble form by induction at low temperature and prevent usage of urea, as using urea will denature the protein and prevent it from binding of the protein to the GST column.Binding is most effective in near-neutral buffers such as Tris-buffered saline (TBS) pH 7.5.

Dilution with PBS in 1:8 need further concentrating steps

It is better to use 1.5 M urea and no dilution

If you cant find condition for soluble expression of your protein i would suggest that you switch to a His-tag and use IMAC purification. The reason is that you can (1) use inclusion body purification protocols which already yields a high purity and (2) IMAC is compatible with 8 M urea and you can refold your protein on the column. This has several advantages compared to refolding in solution. Nevertheless you would probably have to investigate different conditions and additives.

What E. coli strain you use to make your overproduction?

Sometimes the strain may play a role in allowing to produce soluble proteins.

I suggest to use a BL21ai or BL21 (DE3) plysS E. coli strain, both are used for overexpression of T7 promoter-controlled recombinant proteins. In addition, they both contain a T7RNAP that is controlled by promoters in order to precisely control the time and amount of expression of your recombinant protein. In this way you don't have leaky expression of T7RNAP and, as a consequence leaky expression of recombinant protein, as usually happens.

You can also work on changing the IPTG concentration used. Adding Ethanol (3%) at induction may also help. It stress the cell and force it to make chaperone proteins that prevent the formation of inclusion bodies.

Have you tried expression at low temperatures? Reducing the temperature and increasing the time of expression helps avoid inclusion body formation. However, it is not a guarantee that your protein will be found in soluble fraction. The reason why your solubilized fraction did not bind to the column is probably because GST was denatured by 8M urea. Try to express the protein in soluble fraction so you do not have to handle inc.bodies.

If changing expression conditions does not improve soluble expression of your target, switch to His-tag purification. Solubilization of His-tag inclusion bodies usually do not affect affinity purification. You can use either Ni, Co metal columns.

When we work on the fusion proteins the 1st thing is try to obtain the proteins or one of the proteins in the soluble form by reducing the concentration of the induction source (IPTG or Lactose) and also by lowering the temperature of the shaking incubator after inducing the bugs and keeping the culture there for 18 hours. when I had this problem I used IPTG 0.04 mM and temperature was 17-19 C.

If the problem persists that is your proteins are still in insoluble form then there is the 2nd way. Go to the 8 M urea solubilization and refolding using the conditions you were using for your main single protein. Then after refolding and dialysis try to purify the proteins in a single column by changing the pH of the buffer. select the buffer pH range keeping in the mind the the pH sensitivity of the proteins.

If your one protein gets bind to the column and the other not then collect one protein in the form of flow through that is your one protein partially purified after passing through the column and elute the bound protein by sodium chloride different morality fractions.

Here you will have two purified or partially purified proteins. Check the purity through SDS PAGE. Protein in the soluble form or the one obtained in flow through can be purified using ammonium sulphate precipitation method. Precipitate your protein with ammonium sulphate then solubilize the precipitates in water and desalt the content by dialysis and enjoy the purified protein.

Doing this practice keep in the mind the quantity of the starting material. You will loss the material at every step.once the route is established then the experiments go smoothly.

Good luck

I don't think that expression in inclusion bodies should be a problem. As long as your target protein refolds properly, this can be a good method to get high yields and reduce in vivo cleavage of your fusion protein. However, because you need to use urea to dissolve the inclusion bodies, GST or another fusion protein partner may be problematic. If the GST is not folded properly, it won't bind to the affinity column. If you switch to a His tag, you can get efficient binding to a Ni-NTA column even in the presence of urea, so that may be a better option.