Isolation and Purification of Recombinant Myelin Oligodendrocyte Glycoproteins

MOGtag is a recombinant Myelin Oligodendrocyte Glycoprotein fused with solubilization and purification tags for Experimental Autoimmune Encephalomyelitis studies.

To isolate MOGtag, suspend bacterial cells expressing MOGtag in a lysis buffer and incubate.

Enzymes and detergents in the buffer disrupt membrane lipids and degrade the cell wall.

Sonicate to lyse the cells further, releasing insoluble inclusion bodies containing MOGtag.

Centrifuge to pellet the inclusion bodies. Discard the supernatant, resuspend the pellet in buffer, and incubate.

Add guanidine hydrochloride, then incubate to solubilize MOGtag.

Transfer the soluble proteins onto a charged nickel resin, mix, and incubate with shaking to allow MOGtag to bind to the nickel ions.

Centrifuge to pellet the bound MOGtag. Remove the supernatant, resuspend the pellet in an elution buffer, and incubate with shaking.

Imidazole in the elution buffer releases MOGtag from the resin.

Centrifuge again, collect the purified MOGtag-containing supernatant, and store it for further analysis.

Distribute the cultures evenly amongst 250 milliliter bottles compatible with high speed centrifugation, and keep the bottles on ice from this point forward. Pellet the bacterial cells at 22,000 * g for 15 minutes at 4 degrees Celsius. Resuspend and combine all of the bacterial pellets in a total of 30 milliliters of lysis buffer. Transfer this volume to a round bottom 50-milliliter tube capable of high speed centrifugation.

Place this tube in a 30 degree Celsius water bath for 30 minutes. During the incubation time, shake the tube twice to resuspend the cells. Following incubation, transfer the tube onto ice and sonicate the solution at 20 kilohertz and amplitude 70%, pulsing on for three seconds and off for three seconds for 5 pulses. Sonicate the solution for six total rounds of five pulses, allowing the solution to cool on ice in between rounds.

Next, centrifuge the solution at 24,000 * g for 15 minutes at 4 degrees Celsius, then resuspend the pellet in 30 milliliters of buffer A and incubate the solution at 4 degrees Celsius for three hours. Then, add 17.2 grams of guanidine-HCl to the solution. Incubate the sample on ice for one hour to solubilize the MOG tag protein. To purify the MOG tag protein, transfer the entire volume of solubilized protein to the first tube containing nickel resin. After mixing, place the tube horizontally onto a rocker at 4 degrees Celsius for one hour.

After centrifuging the tube at 4,500 * g for eight minutes at 4 degrees Celsius, transfer the supernatant to the second tube of nickel resin and incubate as before. In the meantime, resuspend the nickel resin in the first tube in 40 milliliters of elution buffer and place the tube horizontally on a rocker at 4 degrees Celsius for five minutes before centrifuging as before. Transfer the supernatant containing the eluted MOG tag protein into a 250 milliliter bottle labeled purified MOG tag protein. Keep this bottle at 4 degrees Celsius.