This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Protein Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.
How can I recover soluble protein from inclusion bodies? (Thread 19840)Q I am working with a horrible protein that is expressed as inclusion bodies in E. coli. SAD protein refolding has been problematic. I've been told that it might be possible to recover proteins with native-like secondary structure from the inclusion bodies using a low concentration of urea.
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. It's a dimer: the monomers are 60 kDa with 5 disulfide bonds. The protein is post-translationally glycosylated at 4 sites and has GPI anchors at its C-termini.
Different constructs (aa mutants, constructs with different N- and C-terminal tags), different vectors, and different E. coli host strains have all produced inclusion bodies (even the construct with an N-terminal signal peptide that directed it to the periplasm).
I have almost two years of experience in protein refolding and can tell you that this protein just does not want to refold.
We tried everything, and I mean everything, to purify the active protein from the misfolded protein. We're currently preparing a yeast expression system, but that will take time. While I'm waiting, I would like to try recovering protein from the inclusion bodies.
A1 During the last six months I've expressed twelve proteins in E. coli using the SUMO expression system (Invitrogen). The standard protocol has been:
Isolate the inclusion bodies
Solubilize in 8 M urea or 6 M GuHCl (2 protein required GuHCl)
Purify with Ni-NTA resin under denaturing conditions
Refold against 3 M urea, 20 mM tris, 1 mM DTT, 150 mM NaCl, pH 8.0 using a normal dialysis chamber
Remove the SUMO tag with SUMO protease.
Six of the proteins were able to refold at 2 M urea but six required 3 M. I have to admit that I don't know whether the actual protein of interest folded or not: I'm using a denatured form for ELISA studies.
A2 a) I've used sarkosyl to purify and/or increase yield of several proteins from inclusion bodies. In one case, though, I was able to purify protein, but the protein did not appear to be active (I am not sure of the reason).
I will be trying to cleave N and/or C terminal amino acids to see if that helps on my problem protein.
b) In Analytical Biochemistry (1993) 210:179-187, Frangioni and Neel describe the solubilization of glutathione S-transferase(GST)-fusions with sarkosyl.
c) In fact, for GST-tagged proteins, sarkosyl is mentioned at the end of the GST handbook from Amersham.
A3 a) I recently had to express 16 mutant proteins and all of them were in the insoluble fraction. (I am using the pQE30 vector from Qiagen, so the yield is high.) I had a lot of trouble refolding these proteins—they are all 28 kDa with 16 disulfide bonds.
I finally tried the Refold-Kit from Novagen (basically an ELISA plate with 96 different buffers for refolding). I found two different combinations of buffers that refolded my protein.
b) With that many cysteines and disulfide bonds, are you using one of the origami-based E. coli strains from Novagen? I had to purify a 13 kDa protein that has 16 cysteines and was able to do it only when I used Rosettagami cells.
