Protein rofolding problem - percipitation (Oct/01/2005 )

Hi all,

I got serious and strange problem regarding refolding.

My protein is not over-expressed with normal BL21 series including DE3, AI, SI but only over-expressed with Rogetta cell. However, over-expressed protein is insoluble (100% in inclusion body).

So far, I tired truncated mutant -24 and -34 from C-terminal and doing -20 from N-term truncation now. -24 & -34 from C-term did not give me soluble proteins at all.

At the same time with truncation, I'm doing refolding but very strange thing was happened.

My protein was denatured with CHES; pH 11.0, 1% laurylsarcosine for 2~3 hrs, centrifuged (15K rpm, 20 min), dialyzed against 20 mM HEPES; pH 7.5, 100 mM NaCl, 5 mM DTT (DTT is used only the first buffer to prevent a misfolding). Dialysis buffer (20 mM HEPES; pH 7.5, 100 mM NaCl) was changed 5 times.

Until I change the buffer 4th times, my protein was still soluble and looks fine, but, after the 5th buffer change it started precipitated. I separated pptn and soluble part by centrifugation and realized the pptn part is my protein by SDS-PAGE. I don't know why my protein was percipitated after 5th buffer change (but not precipitated until 4th change).

To solubilize my protein, I tried various detergent screenings (mixing pptn protein solution and detergents -5 times cmc at final concentration after mixing-) and found Anzergent 3-14 can solubilize my protein almost 100%. Thus, I used 5 times cmc Anzergent 3-14 to solublize my precipitated protein solution and it is solubilized!

However, after I dialyzed the solubilized protein in my buffer (20 mM HEPES;pH 7.5, 140 mM NaCl, and 1 mM DTT), it was precipitated again even with 5X cmc detergent! (Anzergent miscel size is around 30kDa, so it will not be dialyzed out from tube - I used 12kDa dialysis tube- ).

Does anybody know why the precipitation keep happening and any idea to solve this problem?

Thank you

If you are not intending to remove the detergent with dialysis, then you can (should) use detergent in your dialysis buffer. Presumably, although the micele size is 30 KD, the dialysis is removing the detergent. You can prevent this by adding detergent to the dialysis buffer.

Hi
It seems likely that your protein do not refold correctly during the conditions that you tried. There are commercial kits for testing different refolding conditions that you can try. The formation of inclusion bodies is a sign of bad solubility maybe a eukaryotic expression system will facilitate soluble expression. If you disclose more details about the nature of your protein, maybe I can give more precise advice. I doubt that truncation will help on soluble expression, many proteins is dependent on the whole sequence for correct folding.

Good luck

Tomas

Hi Tomas (or Bratt) and all,

I dialyzed my protein against the dialysis buffer containing 0.6 mM of Anzergent and it was not heavly precipitated but not 100% clear. In next trial, I will use 0.8 mM detergent to solubilize my pretein well becuase cmc of Anzergent 3-14 may vary in different solution (between 0.2 ~ 0.4 mM).

My protein is bacterial one related to one kind of glycosyltransferase. pI is 9 and 32kDa and the DNA sequence is very AT reach.

Anyhow, after I solubilize my protein again in the buffer solution containing detergent (20 mM HEPES;pH 7.5, 50 mM NaCl, 1 mM Angergent 3-14), I dialyzed the solubilized protein in the dialysis buffer (20 mM HEPES;pH 7.5, 50 mM NaCl, 0.6 mM Angergent 3-14).

Then, protein was filered with 0.22 um one and load onto MonoS column (buffer: 20 mM HEPES;pH 7.5, 50 mM NaCl, 0.6 mM Angergent 3-14). However, for some reason, my protein did not bind to column (which is supposed to be bound due to pI=9.0)

So, I just tried to MonoQ instead. Actually, protein did bind but it bind to strong and not eluted. I lost all of my protein. I do not understand why my protein bound to MonoQ column so well and dont wanna be eluted. (Maybe laurylsarcosine? but I think it was released during dialysis processes.)

Need any help.

Thank you

Hi
A possible explanation for your ionexchange results is that your protein is part of the detergent micelle so the micelle has a positive inner layer and negative outer layer. In that case it is not strange that it binds to Q and not S. I think you need to refold the protein to keep it soluble without detergents especially if you want to make functional or structural studies of the protein. Start out with a refolding kit to find promising conditions. There are several available on the market, do an internet search.

Good luck