実験的自己免疫性脳脊髄炎研究のためのシンプルで効率的な生産とマウスミエリンオリゴデンドロサイト糖タンパク質の精製

The overall goal of this protocol is to generate protein based on the extracellular domain of myelin oligodendrocytes glycoprotein or MOG, which can be used to induce experimental autoimmune encephalomyelitis or EAE. This protocol makes it possible for any lab to make MOG protein in order to use as an antigen to induce EAE. The main advantage of this protocol is that it doesn't rely on specialized protein handling equipment but instead you can use standard equipment found in almost any immunology lab.

Well, this protocol was optimized for MOG tag protein based on the mouse version, it should work for other MOG protein expression systems that incorporate a hestate for purification. Inoculate five milliliters of sterilized broth with the BL 21 MOG tag glycerol stock and incubate it overnight at 37 degrees Celsius and 200 RPM. Add 500 microlitres of 100 milligram per milliliter ampicillin to each of to 500 milliliter flasks containing 500 milliliters of sterile LB.Transfer one milliliter of the overnight culture to each of the two flasks of LB broth.

Incubate at 37 degrees Celsius and 200 RPM for five hours or up to an optical density of 0.6. Once the desired cell density is reached, add 0.5 milliliters of one molar IPTG to each culture flask. Incubate the flask, set 37 degrees Celsius and 200 RPM for four hours and then at room temperature and 75 RPM overnight.

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 times g for 15 minutes at four degrees Celsius. Resuspend and combine all of the bacterial pellets in a total of 30 milliliters of lysate buffer.

Transfer this volume to around bottom 50 milliliter tube capable of high speed centrification. 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, transport the tube on to ice and sonicate the solution at 20 kilohertz and amplitude 70%pulsing on for three seconds and off for three seconds for five pulses, sonicate the solution for six total rounds of five pulses, allowing the solution to cool in ice in between rounds. Next, centrifuge the solution at 24, 000 times g for 15 minutes at four degrees Celsius, then resuspend the pellet in 30 milliliters of buffer A and incubate the solution at four degrees Celsius for three hours. After incubation, sonicate the solution on ice as before, then add 17.2 grams of guanidine HCl to the solution.

Incubate the sample on ice for one hour to solublelize the MOG tag protein. To charge and equilibrate the nickel resin, first wash the resin by adding 40 milliliters of water to each tube containing resin. Lay the tubes horizontally onto a rocker and let them agitate for five minutes at four degrees Celsius.

Once finished, centrifuge the tubes at 4500 times g for eight minutes at four degrees Celsius. Discard the supernatant by pipetting to avoid disturbing the pellet. Then add 40 milliliters of charge buffer to each tube.

Transfer the tubes onto a rocker and let them agitate for 15 minutes at four degrees Celsius before centrifusing done again as before. Discard the supernatant and add 40 milliliters of buffer B to the tubes. Transfer the tubes onto a rocker and let them agitate for five minutes at four degrees Celsius before centrifusing the tubes with resin once more.

To purify the MOG tag protein, transfer the entire volume of solublized protein to the first tube containing nickel resin. After mixing, place the tube horizontally onto a rocker at four degrees Celsius for one hour. After centralfusing the tube at 4500 times g for eight minutes at four degrees Celsius, transfer this 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 evolution buffer and place the tube horizontally on a rocker at four degrees Celsius for five minutes before centrifusing as before. Transfer the supernatant containing the alluded MOG tag protein into a 250 milliliter bottle labeled purified MOG tagged protein. Keep this bottle at four degrees Celsius.

With each elution step, pool the resulting supernatant in this bottle. Add 40 milliliters of strip buffer to the nickel present in the first tube and place horizontally on a rocker for five minutes at four degrees Celsius. Centrifuge the tube, discard the supernatant and recharge the nickel risen as before.

Once finished, move forward with the second tube as was done for the first tube. A total of four rounds of absorption of the solubilized protein onto the charged nickel resin and elution will recover most of the protein. Cut approximately 30 centimeters of snake skin dialysis tubing.

Secure one end with a locking hemostats by folding the end of the snake skin over three times and clamping the folded end with the hemostat. Fill the snake skin with diluted MOG tag protein. Then remove any air bubbles from the snakeskin by forcing them out of the open end.

Finally, seal the other end of the tube using a second locking hemostat. Next, fill a large bucket with one liter of one x acetate buffer with four molar guanidine. Place up to two sections of dialysis tubing containing MOG tag protein into the bucket.

Using tape, secure the hemostats to the side of the bucket to leave room for a magnetic stir bar to spin unhindered in the bottom. Put the bucket in a four degree Celsius room on a magnetic skirt plate and turn it on to a slow rotation rate. The dialysis takes a minimum of three days to gradually reduce the amount of guanidine in the buffer.

Regularly check to make sure that the tube begins intact and that the ends are securely closed. After four to five hours, add one liter of one x acetate buffer to the dialysis bucket. Repeat this process every four to five hours for a total of three times to result in the total of four liters in the bucket.

After discarding half of the buffer in the bucket, refill with one liter of one x acetate buffer and set up the tubing and stir bar. Finally, replace the entire four liter volume in the bucket with four liters of fresh one x acetate buffer and let stir for four to five hours. For best results, do this step on the day of protein concentration.

To concentrate the MOG tag protein, line a pen with aluminum foil and cover the aluminum foil with PEG 3350 and PEG 1000 at a one to one ratio. The PEG 3350 can help prevent protein aggregation during concentration and it's an effective cryopreservative. Put the MOG tag protein containing snakeskin tubing on top of the aluminum foil and cover with PEG 8000.

Let this sit at room temperature and check the volume regularly until the volume is equal to or below the estimated final volume. If the pen becomes over saturated with water during the concentration process, set up a fresh pan with aluminum foil and PEG. Samples of protein taken throughout the purification protocol or run on an SDS page gel to confirm purity.

A representative gel is displayed showing the highly purified MOG tag at 31.86 kilodaltons. To verify that the MOG tag protein has folded correctly, binding of the MOG tag protein to CD19 positive, CD4 negative, nine B cells from lymph nodes from either wild type C57 black six mice or IgH MOG mice that express an immunoglobulin heavy chain specific for MOG protein was assessed using flow cytometry. This protocol requires a minimum of 10 days from beginning to end.

Throughout the isolation procedure is important to keep track of the protein and remember what needs to be kept and what needs to be discarded. Don't forget when using nickel sulfate hexahydrate used in charging the nickel resin, is extremely hazardous and precaution should be taken to prevent inhalation or contact with the skin or eyes. After watching this video, you should have a good understanding of how to produce and purify your own MOG tag protein.

Once purified, MOG tag protein can be emulsified in complete Freund's adjuvant and used as an immunization to induce EAE. EAE induced by MOG tag can be used to study the involvement of multiple cell types such as CD4 or CDAT cells or B cells in central nervous system autoimmunity. Unlike short peptide, which is MHC restricted, protein antigen can be used in any strain of mice.