How to sterilize a protein without denaturating it? - (Apr/16/2006 )
I need to obtain relatively pure band 3 (an integral membrane protein of erythrocytes) for use in cell cultures. I would like to retain as much as possible of the native protein conformation because I´m interested in its antigenic properties. I start with erythrocyte ghosts obtained by hypotonic lysis, then I strip the peripheral proteins with low ionic strength and alkaline pH followed by high ionic strength washes, and finally solubilize the membranes with 1% m/v sodium deoxycholate in 5 mM phosphate buffer with some protease inhibitors.
I posted here a few months ago asking for help on removing the detergent after the aforementioned step, and since I finally got it working, I´m posting my results back so that others might benefit. People suggested me to use dialysis, but this proved to be very tricky. The key point is that free detergent (that is, not protein-bound) with only pass through the membrane if it exists in solution as monomers (low concentration) and not as micelles (high concentration). Since the solubilization conditions usually require high detergent concentrations, the resulting micelles would take weeks to equilibrate across the dialysis membrane. The trick consists in diluting the soluble protein-detergent mixture below the detergent CMC (critical micelle concentration, the detergent concentration above which it only exists as these large aggregates) with a carefully chosen buffer (ionic strength and pH have radical effects on the CMC, so you must do some research on your detergent) and then dialyze as usual, as if you were trying to remove salts, since the dialysis kinetics under these conditions are similar.
In my protocol, after 24 hs dialysis in the cold with 5 buffer changes (each greater than 20 dialysis vols), I concentrate the "detergent-free" protein by ammonium sulfate precipitation at 80% saturation, resuspend the pellet in 1/3 dialysis volume of low ionic strength buffer and perform dialysis again, this time to remove the ammonium sulfate. The precipitated protein slowly goes back into solution as the sulfate goes away, but it never achieves full solubilization. I think this is due to the fact that band 3 is a hydrophobic protein that requires a detergent-solvation layer to stay in solution, since adjusting the ionic strength to a physiological value with 10x PBS doesn´t seem to make a change. Anyways, the insoluble protein doesn´t trouble me much because I´m also planning to use it as a solid antigen, but any tips to bring it back into solution would be great since I would then have both antigen forms, soluble and particulate.
The main issue is that after isolating the detergent-free protein under non-sterile conditions, I need to get it sterile for cell culture. I tried filtering, but the protein aggregates clog the 0.22 um pores and I lose the solid phase. I discarded heat since it is a strong denaturant. I thought of UV irradiation, but I know nothing about it and cannot find any protocols on the subject (intensity? time?). Since it has ionizing effects, could it affect the protein structure? I also considered using acetone or alcohol, but I know from previous attempts that when using high concentrations (80% acetone), the protein gets denatured and will never go back into aqueous solution. Are lower concentrations effective? Can I use other chemicals? What about another method?
Thanks in advance,
Thanks a lot for your informations. This is good to know.
Now, about sterilization. Unfortunately filtration is the best way to sterilize.
If you use UV, you could make some changes, you could make covalent bonds with aromatic groups. I had somewhere a table with the time and energy needed to neutralize various bacteria. unfortunately I'm on vacation right now. PM me if you want me to try to find it next week.
I'm not sure , but I would say you need some time under the UV lamp, and it's heating. you might damage your proteins.
Why don't you add some Na azide in your protein stock, to keep it quite clear, and to avoid degradation by bacteria. Then in your cell media, you have antibiotics, you should not get a contamination.
If someone has a better idea...
Since filtration doesn't work I'd suggest concentration filters like Vivaspin, Centricon or other companies products
Bakteria should act like a giant protein and remain in the tube while your protein should be in the filtrate.
This is just an idea, I never tried this...
and you could use this kind of filtration units regulary to concentrate your protein and/or for buffer exchange, so theres no need for dialysis.