Are you using 8M urea or something else?

The inclusion body protein was probably not fully disaggregated by the detergent, leading to a highly aggregated but soluble material that did not bind to the Ni-NTA column due to the His-tags being inaccessible. GnHCl, a strong chaotrope, was suffcient to disaggregate the protein so that it could bind.

If you want to test this hypothesis, try running the detergent extract and the GnHCl extract through a suitable gel filtration column. Aggregated protein will elute in the void volume and monomeric protein will elute later.

I think Bert van den Berg's group at Newcastle were able to refold a membrane protein from inclusion bodies but that was a bacterial protein. Is this a common thing now? Just add detergent to inclusion bodies and you get perfectly folded membrane proteins? 

I would add GnHCl with the detergents, immobilise on the Ni-NTA and use a refolding protocol or a refolding kit to fold it on the column. Then I would try to see if it were more stable in another detergent and i would iterate. Refolding on the column won't work if the His-tag is hidden from the column in the folded state but I think your protein is a misfolded mess and that's why it doesn't bind directly from the ultra-centrifugation. 

Well Morten, I found your idea of using detergents with GnHCl cocktail is very intriguing. However, I'm also bit worried about the effect of GnHCl or glycerol (refolding cocktail) in fluctuation of the cloud point of the miceller-surfectant.

Hi Abhishek,

From your information it is very clear that there is no problem with His-tag, as it elutes with GnHCl denaturing condition. The problem with detergent arises due to the compatibility of various detergent with Ni-NTA coumn. There is a certain concentaration to which we can use detergents in Ni-NTA column, otherwise it could cause the reduction  of beads hence our protein may not bind to the beads.  I am attaching a pdf describing the compatibility of reagents with Ni-NTA.

hope this helps!

Hello Kanti,

It is very nice of you to attach the compatibility chart. But as per this chart I'm not hitting the maximum limit either to scrape off the functionality of Ni-NTA.

We've had similar issues with His- and other epitope-tagged membrane proteins, where the terminal tag is hidden in the absence of certain detergents. In looking for a workaround, I found the attached Biotechniques approach. Haven't tried it yet, but we will be in the near future. The authors address the basis for atypical re-folding in the article. Perhaps it can help you some. 

Tao et al. (2010) Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques 48(1): 61-64

Is the protein getting into the E. coli membrane or not? If it does you can go for purification from membranes directly. You will need to grow more E. coli but will save a lot of time later.

Alternatively or in combination, how about avoiding inclusion bodies altogether? You can try less IPTG, lower temperature (down to 18C), or addition of 1M NaCl to induce chaperon expression and protein stabilization.

Yoram

Hi Abhisek,

try this protocol to purify your protein from inclusion bodies, it works really well:

http://pubs.acs.org.gate1.inist.fr/doi/pdf/10.1021/bi801729z

good luck!

Hello Emmanuelle,

Will you please send me the pdf, as it appears I supposed to have some kind of security ids to get to this https://auth.inist.fr server.

here it is!