Name: simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies
Uploaded: 2016-10-27T14:00:00
Description: Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

This work was supported by a grant from the Multiple Sclerosis Society of Canada. RWJ is the recipient of the Waugh Family MS Society of Canada Doctoral Studentship Award.

1. Protein Induction
NOTE: In the following steps, BL21 Escherichia coli bacteria transformed with a pET-32 vector containing the sequence for the MOGtag fusion protein (see reference5 and Figure 1) are grown to high densities and are then induced to express the MOGtag protein. See Figure 2 for overall timeline - note that days are approximate and alternate stop points are noted in the protocol. If starting with purified...

<ol>
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Here, we have described a protocol for the production of MOGtag protein and how to generate pure MOG1-125 from the MOGtag protein. This protocol is based both on standard His-tag based protein purification methods, as well as a previously described protocol for the generation of an older MOG-based protein15. Although it is not described here, the primary usage of the MOGtag protein is to induce EAE through immunization with protein antigen. A protocol describing how ...

MS is a human disease characterized by chronic inflammation and neurodegeneration of the CNS which is thought to be driven by an autoimmune response directed towards myelin. The loss of myelin and axons over time result in the gradual decline of cognitive and motor function1. &#34;Experimental Autoimmune Encephalomyelitis&#34; is an umbrella term for animal models of autoimmune disease directed towards CNS myelin. Like human MS, EAE is typically characterized by immune cell infiltration of the CNS and, in some cases, demyelination2. However, the degree to which any given EAE model resembles human MS in part depends on the species or strain used and on the complexity of the underlying anti-myelin autoimmune response.
Anti-myelin autoimmunity can be experimentally induced in several ways, but the most common method used today is to immunize mice with a short peptide of amino acids mimicking the immunodominant CD4+ T cell epitope of a myelin protein. This represents the minimum requirement to induce a pathogenic response. Perhaps the most common of these is a 21 amino acid peptide derived from myelin oligodendrocyte glycoprotein (MOG35-55), which is used to induce EAE in C57Bl/6 mice3. However, for some experimental purposes it is desirable or even necessary to immunize with larger protein antigens and indeed there are several advantages to this over immunization with short peptide. First, because of MHC restriction, short peptides are usually only effective in a very limited range of strains, while larger protein antigens representing either the whole protein or a specific domain can be processed normally for presentation in multiple inbred mouse strains or even in different species4. Second, a larger protein antigen is capable of inducing a more complex immune response incorporating more types of lymphocytes in antigen recognition, rather than limiting antigen recognition to CD4+ T cells. For example, B cells via their B cell receptor (BCR) interact directly with whole rather than processed protein. We and others have shown that B cells activated by MOG35-55 immunization do not recognize MOG protein5. Since B cells were recently demonstrated to play a pathogenic role in human MS6, EAE models that incorporate B cells in autoimmune pathology are increasingly important.
Despite the advantages of using larger protein antigens to induce EAE, there remain few commercially available sources for such proteins. Indeed, while short peptides like MOG35-55 can be synthesized very quickly and at a relatively low cost, the commercial options for MOG protein are limited and cost substantially more to purchase. Nonetheless, there are several expression vectors available for research groups to generate MOG extracellular domain (MOG1-125) themselves. However, all of the expression systems that we have identified in the literature are based on older technologies that have since been replaced with more efficient expression systems7. Further, most are based on rat or human MOG8. For some investigations of autoimmunity in mice, an antigen based on the mouse MOG autoantigen is preferable. Finally, all MOG-based proteins that we have identified, either commercial or as expression vectors, are fusion proteins containing additional amino acids to the MOG1-125 base. These include a tag for purification and usually other sequences as well, many of which with a function we were unable to identify.
To address these limitations, we generated a novel fusion protein based on the mouse MOG extracellular domain fused to a tag containing thioredoxin to combat the known insolubility of MOG protein5. The tag sequence also contains a 6xHis sequence for purification and a TEV protease cleavage site that allows for the complete removal of all tag sequences, if desired. This is the only method that we are aware of to generate pure MOG1-125 protein. To facilitate production of large amounts of protein, the MOG1-125 sequence was codon-optimized for bacterial expression and the MOGtag fusion protein was inserted into the pET-32 expression system. Here, we describe in detail the protocol to produce and purify MOGtag protein, and pure MOG1-125, using non-specialized equipment available to most immunology laboratories.

<table border="0" cellpadding="0" cellspacing="0" width="1434">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">BL21 E.coli- pet32-MOGtag</td>
			<td style="width:139px;">Kerfoot lab</td>
			<td style="width:119px;"></td>
			<td style="width:803px;">These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LB broth miller</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LBL407.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Ampicillin</td>
			<td style="width:139px;">bio basic</td>
			<td style="width:119px;">AB0028</td>
			<td>Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">IPTG</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IPT002.5</td>
			<td>Reconsititute the powder into H2O at 1M and store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Chicken-egg lysozyme</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LYS702.10</td>
			<td>Reconstitute in H2O at 50 mg/ml and store at -80 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Triton-X100</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">T-8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Phosphate buffered saline</td>
			<td style="width:139px;">life technologies</td>
			<td style="width:119px;">20012-027</td>
			<td>Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium chloride</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SOD004.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS002.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Imidazole</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IMD508.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Guanidine-HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">G3272</td>
			<td>The quality must be greater than 98% purity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.5M EDTA</td>
			<td style="width:139px;">bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nickel (II) sulfate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">NIC700.500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">His bind resin</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">69670-3</td>
			<td>Store in 20% ethanol 80% H2O at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Anhydrous ethanol</td>
			<td style="width:139px;">Commercial Alcohols</td>
			<td style="width:119px;">P016EAAN</td>
			<td>Dilute with water as needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">ACE222.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium acetate trihydrate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SAA305.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bovine serum albumin standard</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Bio-rad protein assay dye reagent concentrate</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Ethylenediamine tetraacetic acid, disodium salt dihydrate</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-base</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">7000 MW Snakeskin dialysis tubing</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">68700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">2-mercaptoethanol</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M3148-25ml</td>
			<td>This reagent should not be handled outside of a fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">AcTEV protease</td>
			<td style="width:139px;">lifetechnologies</td>
			<td style="width:119px;">12575-015</td>
			<td>Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Polyethyleneglycol 3350</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG335.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">polyethyleneglycol 8000</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG800.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nunc MaxiSorp flat-bottom 96 well plate</td>
			<td style="width:139px;">ebioscience</td>
			<td style="width:119px;">44-2404-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sonicator</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">FB-120-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eon microplate spectrometer</td>
			<td style="width:139px;">Biotek</td>
			<td style="width:119px;">11-120-611</td>
			<td>This equipment uses the Gen5 data analysis software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gen5 data analysis software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">sodium dodecyl sulphate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SDS001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bromophenol blue</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">BRO777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycerol</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLY001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein desalting columns</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">89849</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycine</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLN001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">precast 12% polyacrylamide gel</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">456-1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rapid stain reagent</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">553215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gel dock EZ imager</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">White Light Sample Tray</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708272&#160;</td>
			<td>Used along with gel dock EZ imager for coomassie blue stains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein ladder</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1610375</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

Once the purification is complete, samples collected in steps 1.4, 2.1, 3.4, and the final product from step 6.4 should be run on a protein gel (Figure 3A). MOGtag should first appear as a 31.86 kDa band in the TO/N sample, but not T0, and should be the only band in the final pure product. To test whether the MOGtag protein has correctly folded, the MOGtag protein can be used to label MOG-protein specific B cells by F...

Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

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.

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