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Pros and Cons of refolding proteins

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#1 Smog187



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Posted 29 July 2016 - 12:36 PM

Hi everyone, 


I've been trying to solubly express my protein in e. coli for the last year and a half. I have created fragments and tried them, and those themselves have problems. My protein is about a 1000 amino acids (112kDa) and always goes to the pellet no matter what I do. I'm pretty sure it's going into the inclusion bodies, as I have tried membrane preps with a few different detergents and it still goes into the pellet. I also fused my protein to MBP, but after cleavage my protein fell out of solution. 


Recently I resuspended my pellet in 6M urea and was able to extract my protein from the pellet, which got me thinking of trying to refold my protein. I know this is also a long arduous process, and before I attempt it my biggest fear is what if I get a incorrectly folded protein? I don't want to do a bunch of experiments only to find out it was misfolded. This scares me because to date, no one has purified this protein so I have no way of knowing if it is correctly folded. I would follow up the refolded protein my CD, and maybe crystallography. 


Thanks for any advice. 


#2 mdfenko


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Posted 01 August 2016 - 04:25 AM

the safest way to refold is to dialyze against reduced urea in small steps.


if the protein is an enzyme then you should be able to determine if it's folded correctly by assay.


most of the misfolded protein will probably precipitate during dialysis.

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#3 labtastic



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Posted 01 August 2016 - 12:05 PM

Sounds like an ugly protein you have there.


Unfortunately, the only way to confidently say your protein is refolded to its native state is with some sort of functional assay.


My favorite way to re-fold proteins is, if your protein is his-tagged and you have an FPLC, to bind solubilized inclusion bodies to nickel resin, wash resin with 6M guanidine, then do a slow (overnight) linear gradient from 6M to 0M guanidine while protein is on column. Elute re-folded protein with traditional elution buffer. Then I would gel-filter the protein and pool only the peak that elutes at the expected molecular weight (ie not aggregated protein).


One other suggestion that is to express a functionally identical protein from a different organism (or what you think is a functionally identical based on sequence alignments, phylogeny, conserved residues, etc) and is 70-90% identical in sequence. I have seen many examples where one ortholog expresses like garbage (inclusion bodies, low yield, all aggregated, crashes out, etc), but then a different ortholog with same functionality and 80% identical expresses and behaves like a dream. 

#4 Nancy.Lee



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Posted 10 October 2016 - 10:39 PM

 In order to obtain biologically active and soluble protein in high yield, inclusion bodies must then be solubilized and refolded in vitro. The following links about protein refolding may help you:




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