If the protein does not have to be correctly folded for the vaccine work, replace the urea with an ionic detergent using dialysis. That should keep the protein in solution. Remove most of the detergent by dialysis, leaving just enough to keep the protein from precipitating. There is probably some low limit on how much detergent you can inject without harm. Some experimentation with different detergents may be needed.

In the past, I found that a beta-barrel type membrane protein purified in urea could be exchanged by this method into an anionic detergent called TOPPS, which could be almost completely removed without the protein then precipitating, presumably because the detergent stuck tightly to the hydrophobic parts of the protein.

Purified P-glycoprotein, a polytopic alpha-helical membrane protein, could be exchanged from 6 mM Zwittergent 3-12 into 2 mM CHAPS, a zwitterionic detergent, a concentration below its CMC, and the protein remained soluble.

In what buffer are you eluting from the ProBond resin?

As far as I know the 'solution' for your problem is to use high concentrations of urea.

See for example the outer membrane protein proOmpA:

https://onlinelibrary.wiley.com/doi/abs/10.1002/j.1460-2075.1988.tb03015.x

Or the outer membrane prePhoE:

http://www.jbc.org/content/264/35/20827.full.pdf

Perhaps this helps you as well.

Eluted using the denaturing buffer system. Final buffer: 8 M Urea, 20 mM Sodium Phosphate, pH 4.0 500 mM NaCl

Does your protein contains cysteines?

Then DTT might do the 'trick'.

Your solution has extremely low ionic strength and very little buffering capacity. Try increasing the Tris concentration to 50 mM and adding 100-200 mM NaCl.

Triton X-100 and Tween-20 are not great choices because they are subject to oxidation. Consider using dodecylmaltoside, n-octylglucoside, or CHAPS.

It would probably help to say that the proteins are in solution after purification, however they precipitate during dialysis. I need to remove the Urea as it is not conducive to vaccine studies. The protein sequence has one cysteine in it.

If the protein does not have to be correctly folded for the vaccine work, replace the urea with an ionic detergent using dialysis. That should keep the protein in solution. Remove most of the detergent by dialysis, leaving just enough to keep the protein from precipitating. There is probably some low limit on how much detergent you can inject without harm. Some experimentation with different detergents may be needed.

In the past, I found that a beta-barrel type membrane protein purified in urea could be exchanged by this method into an anionic detergent called TOPPS, which could be almost completely removed without the protein then precipitating, presumably because the detergent stuck tightly to the hydrophobic parts of the protein.

Purified P-glycoprotein, a polytopic alpha-helical membrane protein, could be exchanged from 6 mM Zwittergent 3-12 into 2 mM CHAPS, a zwitterionic detergent, a concentration below its CMC, and the protein remained soluble.

Dear Logan

so if i understood well, did you extract and purified the protein with urea and than you tried to replace urea with triton just because urea is not suitable for immunitation. it is correct?

did you think to test detergents more used for menbrane proteins as ddm, dm or og?

when you refer to hydrophylic outer membrane, it is a beta barrel membrane protein simar to ompa or it is a alpha helix transmembrane similar to gpcr?

ciao

Manuele

Manuele Martinelli, the structure has not been solved yet, however, from predictive modeling it is an alpha helix transmembrane protein.

If it is intended to be used as an antigen, try including different concentrations of arginine (in the 0.5 - 2 M range) in the renaturation buffer. That guanidinium group in the side chain helps keep things in solution and it is my understanding that arginine can be used as buffer additive for systemic administration to animals/humans. Also, it is important to include a reducing agent as suggested by Rob Kellerto get rid of any pesky disulfides. You may also try including a reduced/oxidized glutathione pair (it works wonders, although it is expensive).

Also, you try renaturing by dilution (add slowly the protein to a 1,000- to 10,000 larger volume of renaturing buffer under constant mixing). Chances for aggregation are plenty inside a dialysis bag but less so when diluted. You can later concentrate the protein (if it does not precipitate) with a strong ion exchanger.

If I were you and there weren't any T-cell epitops worth keeping in the transmembrane portion, I would also consider making an expression construct without it. That transmembrane helix will bring nothing but trouble...

Hope it helps!

DEar Logan

i think that you strategy, unfolding with urea and renaturation with detergent could be appropriate for beta barrel transmembrane proteins which has a very stable fold, but if your protein is an alpha helix transmembrane protein, i think that you have to resuspend directly the protein in the right detergent and you cannot use triton (that could be usefull for small scale trials to determine the expression of the protein) but you have to use detergents ( DM, DDM, OG) that has micelle size more compatible with proteins and maintain the detergent during the entire purification because with urea you destroid the protein folding and refold a TM protein is not obvious, you need to use something able to extract the protein in a gentle way.

You can find an example here:

Article A Versatile Strategy for Production of Membrane Proteins wit...

Unfortunately some detergents are quite expensive and exchange one with the other is quite difficult, but i think that if you would increase your probability to succeed you have to perform a trials in this direction.

good luck

Manuele

From personal experience, the zwitterionic sulfobetaine detergent (C14 length) performed best at preventing aggregation (during purification of a bacterial cotransporter) from many nondenaturing detergents (nonionic and zwitterionic) that I screened. (aka: SB 3-14 and Zwittergent 3-14).

See also: nondetergent sulfobetaines remarkably stabilize proteins against aggregation (Anal Biochem. 352 (2006) 299–301).

Hi , I agree with Manuele Martinelli answer