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 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.
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. "Experimental Autoimmune Encephalomyelitis" 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.
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 pET-32 MOGtag vector DNA, it will be necessary to chemically transform it into competent BL21 E. coli bacteria using ampicillin selection, as has been well-described9. Successful transformation can be confirmed by purifying DNA from selected bacteria using a standard commercial kit, followed by digestion with the restriction enzymes Age1 and Sac1 to produce a 424 bp band on an agarose gel10.
2. Harvesting MOGtag Protein
NOTE: At this stage, the bacteria will have produced large quantities of MOGtag protein. To harvest MOGtag, bacteria are first lysed in a Triton X-100 buffer followed by sonication. MOGtag is then released from inclusion bodies and denatured with imidazole and guanidine, resulting in a crude protein solution containing the MOGtag protein.
3. Protein Purification
NOTE: In the following steps the MOGtag protein will be purified through 4 rounds of absorption onto charged nickel resin (via the His-tag) and elution.
4. Measuring Protein Concentration
NOTE: Before proceeding further it is necessary to quantify the amount of purified MOGtag protein generated in section 3. This value will be used to determine the final volume to concentrate the protein to at the end of the protocol. We describe a standard Bradford Assay here. The concentration of purified MOGtag protein is determined by comparing the spectral absorbance of serially diluted MOGtag protein to a standard curve of bovine serum albumin (BSA) at a known concentration.
5. Dialysis
NOTE: Dialysis is performed to gradually remove guanidine from the solution containing purified, denatured MOGtag to allow the protein to refold. Care must be taken during this step as MOG itself is very insoluble, and while this is improved by the presence of the thioredoxin tag, it is still prone to come out of solution. Therefore, refolding should be performed gradually and at a relatively low MOGtag concentration.
6. Concentrating MOGtag Protein
NOTE: In the final step, refolded MOGtag protein is concentrated to the working dilution for storage. As MOGtag is very insoluble, it should not exceed 5 mg/ml. This concentration is approximately equimolar with 0.4 mg/ml MOG35-55 peptide, which is commonly used to induce EAE in mice (mixed 1:1 with complete Freund's adjuvant (CFA)). During the concentration process it is not uncommon for a small amount of protein to come out of solution in the form of white precipitate. Excessive precipitation is a problem, however.
7. Generating MOG1-125 from MOGtag Using TEV Protease (Optional)
NOTE: This optional step continues from the end of step 4. If MOG1-125 without any extra tag sequences is required, the tag sequences can be removed using TEV protease (Figure 4). As far as we are aware, there is no other expression system capable of generating pure MOG1-125. However, it should be noted that without the thioredoxin tag, MOG1-125 is highly insoluble and this may cause problems during purification and handling, and for this reason remove the tag if absolutely necessary for experimental reasons. Several steps are required to generate pure MOG1-125. MOGtag is first dialyzed into TEV protease cleavage buffer. Following digestion with TEV protease, the volume is reduced to aid with later purification steps, then dialyzed into buffer B, and then the His-tag containing tag sequence is removed using nickel resin. Finally, protein is quantified and pure MOG1-125 is concentrated to the final concentration.
8. SDS-PAGE Gel to Confirm MOGtag Production and Purity
NOTE: Samples taken from steps 1.4, 2.1, 3.4, and 6.4 are analyzed by standard SDS-PAGE to confirm MOGtag production and purity. This step should be performed after the final purification of either MOGtag or MOG1-125.
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 FACS. By labeling mouse lymphocytes with a 1:1,000 dilution of MOGtag along with a secondary antibody directed against the His-tag and a fluorescently-labelled tertiary anti-IgG1 antibody, MOG-specific B cells can be identified (Figure 3B). Alternatively, MOGtag can be directly conjugated to fluorophores to reduce background staining (resulting from B cells binding to the secondary and tertiary antibodies) as described in reference5 to identify MOG-specific B cells. This is necessary when trying to identify MOG-binding B cells in wild type mice, as these cells are normally very rare5. IgHMOG mice have a heavy chain knockin that, when paired with an appropriate kappa light chain, confers specificity for MOG. As the binding of MOGtag is enhanced amongst the IgHMOG B cells, this confirms that this protocol generates a properly folded antigen. Importantly, IgHMOG B cells contribute to autoimmune pathology in models of MOG-directed EAE12, confirming that MOGtag induces both T and B cell autoimmunity.
Before starting dialysis to refold the protein as described in section 5, it is necessary to measure the protein concentration using a Bradford assay, as described in section 4. Representative results of a Bradford assay are shown in Table 1 and summarized in Figure 5.
The generation of pure MOG1-125 is accomplished by the addition of TEV protease to MOGtag protein ultimately resulting in the cleavage of the MOGtag protein into MOG1-125 and tag sequence as shown in Figure 6. Subsequent nickel resin purification removes MOGtag, tag sequence, and TEV protease impurities ultimately resulting in pure MOG1-125 as shown in Figure 6.
Figure 1: MOGtag protein. (Duplicated here with permission from Dang et. al.5). Linear structure, and amino acid and DNA sequences of the MOGtag fusion protein. The DNA sequence for the extracellular domain of mouse MOG (MOG1-125, lower sequence in blue) was codon-optimized for expression in bacteria (black). This sequence was synthesized and inserted into a vector to create an N-terminal fusion to a tag containing thioredoxin and an S-Tag to counteract the known insolubility of the MOG protein13,14, as well as a 6x His Tag for purification15. A TEV protease cleavage site separates the MOG1-125 from the tag sequences. TEV-mediated cleavage between glutamine-164 and glycine-165 using an alternative consensus TEV cleavage site16 results in removal of all non-MOG amino acids. Please click here to view a larger version of this figure.
Figure 2: Overview of the steps required to produce pure MOGtag protein. To generate MOGtag protein, bacteria expressing the MOGtag protein are grown to high densities then induced to express MOGtag using IPTG as listed in section 1. After an overnight culture, the bacteria are lysed and through a series of pelleting steps the protein fraction containing inclusion bodies, which contains MOGtag, is extracted as listed in section 2. MOGtag is then purified from the crude protein fraction through four cycles of absorption onto charged nickel resin and elution of the MOGtag protein as listed in section 3. A portion of the pooled eluate is then taken for a Bradford assay to determine the yield of MOGtag protein and the rest of the eluate is dialyzed into acetate buffer over the course of several days as listed in sections 4 and 5. Lastly, the protein is concentrated using PEG 3350 and PEG 8000 to a final concentration of 5 mg/ml based upon the yield of MOGtag determined in the Bradford assay as listed in section 6. The entire process can take a minimum of 10 days, with the start day for each step shown in brackets. However, alternative stop and start points are listed in the protocol, and steps can be spread over a greater amount of time if desired. Please click here to view a larger version of this figure.
Figure 3: Purification of the MOGtag protein and assessment of its activity. (A) Shown are protein samples that were collected from various points across the protein purification procedure and run on a SDS-PAGE gel. T0= BL21 bacteria prior to protein induction (collected in step 1.4), TO/N= BL21 bacteria post-induction of protein expression (collected in step 2.1), Crude MOGtag= Solubilized MOGtag protein prior to protein purification (collected in step 3.4), Pure MOGtag= MOGtag protein after purification (collected in step 6.4). (B) Binding of the MOGtag protein to CD19pos CD4neg naive B cells from lymph nodes from either wild type C57Bl/6 mice or IgHMOG mice that express an immunoglobulin heavy chain specific for MOG protein7,17 was assessed using flow cytometry. MOGtag-specific B cells were identified by staining lymph node cells with MOGtag followed by a secondary anti-his tag antibody and a fluorescent tertiary anti-IgG1 antibody. Staining of cells from C57BL/6 or IgHMOG mice is shown along with a MOGtag fluorescence minus one (FMO) control stain of IgHMOG cells. Please click here to view a larger version of this figure.
Figure 4: Overview of the steps to generate MOG1-125 from MOGtag protein. After collecting the MOGtag eluate as described in step 3.5 of the protocol, the MOGtag protein is dialyzed into TEV protease cleavage buffer. Once the dialysis is complete, TEV protease is added to the MOGtag solution resulting in the cleavage of MOGtag into the MOG1-125 protein. The volume of the cleavage solution is then reduced and dialyzed into buffer B prior to protein purification. Impurities from the cleavage solution are extracted through four successive rounds of absorption onto charged nickel resin and elution of the impurities ultimately resulting in a solution of pure MOG1-125. The concentration of the MOG1-125 protein is determined through a Bradford assay and the protein is folded over the course of several days through dialysis. Once dialysis is complete, the MOG1-125 protein is concentrated to 2.24 mg/ml using PEG 3350 and PEG 8000. This protocol is discussed in detail in section 7. Please click here to view a larger version of this figure.
Figure 5: Example of a Bradford assay standard curve for determining the concentration of MOGtag protein. BSA standard readings taken from Table 1 were plotted to obtain a linear regression formula for calculating the MOGtag concentration based upon the optical density at 595 nm. Please click here to view a larger version of this figure.
Figure 6: TEV cleavage of MOGtag protein and subsequent purification of MOG1-125. Shown are protein samples run on an SDS-PAGE gel demonstrating purification of MOG1-125. Pure MOGtag = MOGtag protein prior to TEV cleavage (collected in step 6.4), MOGtag w/ TEV = Protein fraction that was collected after 72 hr of incubation of MOGtag with TEV protease (collected in step 7.5), Elution = Protein fraction that remained bound to the nickel resin during the MOG1-125 purification protocol (collected in step 7.9), Pure MOG1-125 = MOG1-125 protein after purification (collected in step 7.12). Please click here to view a larger version of this figure.
BSA (mg/ml) | 0.4 | 0.35 | 0.3 | 0.25 | 0.2 | 0.15 | 0.1 | 0.05 | 0 |
1.079 | 0.998 | 0.948 | 0.853 | 0.769 | 0.699 | 0.583 | 0.493 | 0.373 | |
1.071 | 1.014 | 0.95 | 0.854 | 0.777 | 0.681 | 0.579 | 0.484 | 0.375 | |
1.069 | 1.017 | 0.944 | 0.848 | 0.781 | 0.687 | 0.592 | 0.494 | 0.374 | |
MOGtag dilution | 1 | 1/5 | 1/25 | ||||||
1.327 | 1.013 | 0.493 | |||||||
1.332 | 1.063 | 0.491 | |||||||
1.367 | 1.088 | 0.488 |
Table 1: Representative values from a Bradford assay for determining the concentration of MOGtag protein. BSA dilutions at known concentrations are used for determining the standard curve shown in Figure 5. The dilutions of MOGtag protein post purification are used to determine the final MOGtag protein concentration. Rows contain the optical density measured at 595nm and each row represents one replicate of a total of 3 replicates at each indicated concentration.
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 EAE is induced in mice, which is compatible with the MOGtag protein, can be found in reference3. We have previously demonstrated that immunization with MOGtag or MOG1-125 derived from MOGtag not only induces CNS autoimmune disease with greater spinal cord inflammation and demyelination compared to the standard MOG35-55 peptide, but also that pathogenic IgHMOG B cells that recognize MOG protein are activated to produce a germinal center response in response to MOGtag or MOG1-125, but not to MOG35-555. Therefore, immunization with MOGtag does indeed induce an appropriate anti-MOG B cell response.
MOGtag protein is purified through absorption onto charged nickel resin (via the His-tag) and elution. Because of the large quantity of protein generated in the previous steps, multiple rounds of absorption and elution are required to collect most of the protein. We have found that at least 4 rounds are required to isolate the majority of protein if using an appropriate volume of resin for 50 ml tubes. If desired, the protocol could be scaled up to use more or larger tubes and more nickel resin to reduce the number of rounds of absorption. Alternatively, high performance liquid chromatography (HPLC) with nickel columns can effectively purify his-tagged proteins18 and indeed we have found that HPLC can efficiently purify MOGtag protein (unpublished observations). As HPLC is not accessible to many standard immunology labs, the protocol listed here is designed to be performed using common lab equipment. In addition to his-tag purification, the MOGtag protein does contain an S-tag that is compatible with S-tag purification protocols if preferred14.
Recombinant proteins based on MOG (mostly human or rat) have been described previously8. These were based on older expression systems that have since been replaced by systems driven by stronger transcriptional promoters capable of producing larger quantities of protein in Escherichia coli bacteria. Our MOGtag expression system uses the efficient T7 promoter in the pET-32a(+) vector, which is significantly more efficient than systems available for in-house production of MOG protein19. However, it should be noted that the MOGtag protein described here is based on mouse MOG. Immunization with human MOG protein has been shown to induce EAE in mice that has different features compared to disease induced using murine MOG8. Further, rat MOG may be more immunogenic than mouse MOG in mice in some cases20. Therefore, for some experimental purposes mouse-based MOGtag may not be ideal. We are in the process of generating several different version of the MOGtag protein than may suit some purposes better, and the purification protocol described here will work for all of these. In the meantime, it is possible that the purification protocol described here may work for other MOG expression systems, or even other proteins, as long as they incorporate a 6x His Tag for absorption to nickel resin. However, without the thioredoxin tag, solubility of MOG protein at higher concentrations may be an issue, and of course it will not be possible to generate pure MOG1-125 as this is unique to the MOGtag system.
Measuring the MOGtag protein concentration prior to protein refolding via dialysis is essential to the success of this purification protocol. If the concentration is too high, the protein may aggregate and fall out of solution instead of folding into individual proteins. This can be seen during the dialysis protocol as white precipitate forming at the bottom of the dialysis tubing. If this is occurring, dissolve the MOGtag protein again with 6 M guanidine and measure the protein concentration. Dilute the protein to 0.5 mg/ml then start dialysis again as no precipitates should form at 0.5 mg/ml. For MOG1-125, precipitates can be expected to form as the protein is highly insoluble. We have found that this will not affect the folding of MOG1-125 provided that the protein was adequately diluted prior to dialysis.
For the TEV protease to effectively cleave the MOGtag protein into pure MOG1-125 it is important to use a sufficient amount of TEV protease. Older versions of TEV proteases are inactivated through self-cleavage21 however more modern versions suffer from insolubility issues22 thus it is important to add enough TEV protease to achieve sufficient cleavage of the MOGtag protein before TEV protease inactivation. Since commercially available TEV protease can be costly, we recommend producing and purifying TEV protease as described in reference22. When using pure mouse MOG1-125 protein for experiments, it is important to note that the protein is highly insoluble and may precipitate out of solution, and therefore must be mixed extensively prior to use.
In summary, here we have described a simple protocol for producing and purifying large quantities of MOGtag protein. Furthermore, the addition of a TEV protease cleavage site to our MOGtag protein provides the opportunity to generate pure MOG1-125 if needed. MOGtag protein is recognized and bound by anti-myelin autoimmune B cells and MOGtag-induced EAE incorporates B cell-mediated pathology5. Therefore, MOGtag protein overcomes the major hurdles limiting the wide-spread use of protein antigen to induce EAE.
The authors have nothing to disclose.
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.
BL21 E.coli– pet32-MOGtag | Kerfoot lab | These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request. | |
LB broth miller | Bioshop | LBL407.1 | |
Ampicillin | bio basic | AB0028 | Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 °C. |
IPTG | Bioshop | IPT002.5 | Reconsititute the powder into H2O at 1M and store at -20 °C. |
Chicken-egg lysozyme | Bioshop | LYS702.10 | Reconstitute in H2O at 50 mg/ml and store at -80 °C. |
Triton-X100 | Sigma | T-8532 | |
Phosphate buffered saline | life technologies | 20012-027 | Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient. |
Sodium chloride | Bioshop | SOD004.1 | |
Tris-HCl | Bioshop | TRS002.1 | |
Imidazole | Bioshop | IMD508.100 | |
Guanidine-HCl | Sigma | G3272 | The quality must be greater than 98% purity. |
0.5M EDTA | bioshop | EDT111.500 | |
Nickel (II) sulfate | Bioshop | NIC700.500 | |
His bind resin | EMD Millipore | 69670-3 | Store in 20% ethanol 80% H2O at 4 °C |
Anhydrous ethanol | Commercial Alcohols | P016EAAN | Dilute with water as needed. |
Glacial acetic acid | Bioshop | ACE222.1 | |
Sodium acetate trihydrate | Bioshop | SAA305.500 | |
bovine serum albumin standard | bio-rad | 500-0206 | |
Bio-rad protein assay dye reagent concentrate | bio-rad | 500-0006 | |
Ethylenediamine tetraacetic acid, disodium salt dihydrate | Fisher scientific | BP120-500 | |
Tris-base | Bioshop | TRS001.1 | |
7000 MW Snakeskin dialysis tubing | Thermoscientific | 68700 | |
2-mercaptoethanol | Sigma | M3148-25ml | This reagent should not be handled outside of a fume hood. |
AcTEV protease | lifetechnologies | 12575-015 | Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17 |
Polyethyleneglycol 3350 | Bioshop | PEG335.1 | |
polyethyleneglycol 8000 | Bioshop | PEG800.1 | |
Nunc MaxiSorp flat-bottom 96 well plate | ebioscience | 44-2404-21 | |
Sonicator | Fisher scientific | FB-120-110 | |
Eon microplate spectrometer | Biotek | 11-120-611 | This equipment uses the Gen5 data analysis software. |
Gen5 data analysis software | BioTek | ||
sodium dodecyl sulphate | Bioshop | SDS001 | |
bromophenol blue | Bioshop | BRO777 | |
Glycerol | Bioshop | GLY001 | |
Protein desalting columns | Thermoscientific | 89849 | |
Glycine | Bioshop | GLN001 | |
precast 12% polyacrylamide gel | bio-rad | 456-1045 | |
Rapid stain reagent | EMD Millipore | 553215 | |
Gel dock EZ imager | bio-rad | 1708270 | |
White Light Sample Tray | bio-rad | 1708272 | Used along with gel dock EZ imager for coomassie blue stains |
Protein ladder | bio-rad | 1610375 |