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

Summary

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.

Abstract

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 (MOG_tag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG_1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOG_tag or MOG_1-125. This protocol consistently generates over 200 mg of MOG_tag protein. Immunization with either MOG_tag or MOG_1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

Introduction

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 a....

Protocol

1. Protein Induction

NOTE: In the following steps, BL21 Escherichia coli bacteria transformed with a pET-32 vector containing the sequence for the MOG_tag fusion protein (see reference⁵ and Figure 1) are grown to high densities and are then induced to express the MOG_tag 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 MOG_tag vector DNA, it will be necessary to chemically transform it into competent BL21 E. coli bacteria using ampicillin s....

Results

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). MOG_tag should first appear as a 31.86 kDa band in the T_O/N sample, but not T₀, and should be the only band in the final pure product. To test whether the MOG_tag protein has correctly folded, the MOG_tag protein can be used to label MOG-protein specific B cells by F.......

Discussion

Here, we have described a protocol for the production of MOG_tag protein and how to generate pure MOG_1-125 from the MOG_tag 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 MOG_tag protein is to induce EAE through immunization with protein antigen. A protocol describing how .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.
0.5 M 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