I´ve been trying to solubilize proteins from E. coli inclusion bodies using several methods. The last one that I´ve tried was 6M Guanidine. However it seems that it did not help.
I would like if I should use 8M Urea in order to obtain better results.
Thanks in advance for your help!
Guanidine hydrochloride is about 1.5-2 x more potent as a chaotrope than urea. The strength of the chaotrope needed to solubilize inclusion bodies varies from protein to protein. I prefer to run small scale tests to gauge the minimal strength of chaotrope needed, as it may improve the prospect of renaturing the protein by not destroying some of the secondary or tertiary structure elements that may be present in the insoluble protein. I prefer to start with urea because it is non ionic and doesn't mess up the gel pattern on SDS-PAGE.
Hello Rafael, I hope the following article can help with your problem.
Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli.
Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LC, Panda AK.
The inclusion body wasn't solubilezed at all or somehow disolved a little? I have tried to solubilize inclusion body using 6M Guanidine and it worked very well and 90% IB was disolved but still have a little that is insoluble. I mean you don't need concern too much for the solubility of IB if you can get sufficient protein for downstream application. One more suggestion is that it would be better to make 6M Guanidine in refolding buffer if you will perform refolding next step.
8M urea is perfect. first you can dissolve your inclusion bodies in 5 M urea for 3 hours. it removes many less hydrophobic proteins from your inclusion. now centrifuge them back and redissolve in 8M urea.
If 8M urea does not work, I´d suggest to try 7M urea, 2M thiourea, 4% CHAPS in 50mM Tris pH 8. This buffer composition is better suited for solubilization of hydrophobic proteins than using urea alone. Additionally, if your protein contains many cysteine residues, it may help to add 1% (w/v) DTT to reduce disulfide bridges.
I agree with Birendra. I usually add 4M urea to dissolve the IB + stirring w/ stirbar and then spin down. Then add 8M urea + stirring for overnigght. you are good to go in the next morning
6M GITC will dissovle everything. pass on to the IMAC column, wash the column with Urea gradient (8M-0) in presence of DTT to get the protein into correct folding on the column. then elute
I have successfully used 1% sarcosyl for solubilisation with subsequent refolding by rapid dilution to obtain proteins with defined structure (see attachment). Note that pH during solubilisation is critical and should be around 8.0, reducing agents might be also further increase yields and work best also at higher pHs.
Article Optimisation of expression and purification of the feline an...
An advantage of urea is that it is not ionic, so you could do ion-exchange chromatography on the solubilized inclusion bodies as a purification step. You couldn't do this with guanidine hydrochloride because it is ionic. However, if your protein is solubilized but still aggregated, it probably won't bind to the ion-exchange column. This is a pretty good way of testing whether the solubilization was successful. Make sure you use freshly prepared urea solutions, since it is somewhat unstable.
I dissolved the entire bacterial pellet with 8M urea and then did His purification. Worked very well! The eluted protein was very pure.
Guanidine hydrochloride is about 1.5-2 x more potent as a chaotrope than urea. The strength of the chaotrope needed to solubilize inclusion bodies varies from protein to protein. I prefer to run small scale tests to gauge the minimal strength of chaotrope needed, as it may improve the prospect of renaturing the protein by not destroying some of the secondary or tertiary structure elements that may be present in the insoluble protein. I prefer to start with urea because it is non ionic and doesn't mess up the gel pattern on SDS-PAGE.
For Ion exchange you can use 8 M urea !!!! as denaturing agent, 6 M Gn Hcl did not work for us because of ionic nature.
All the best
I would try to avoid recovering protein from IB, of course the obtained protein can be very pure but completely denatured and effective refolding procedures are difficult to perform. I would better try to change upstream procedures to reduce expression and increase the soluble fraction of the protein or change the plasmid promotor to obtain less protein but of better quality. In any case, it would depend on the posterior usage of your protein, you always should have this issue into account.
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