Isolation and Purification of Recombinant Myelin Oligodendrocyte Glycoproteins

Abstract

Source: Jain, R. W., et al. Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies. J. Vis. Exp. (2016)

This video demonstrates the isolation and purification of MOGtag, a recombinant Myelin Oligodendrocyte Glycoprotein (MOG) used in Experimental Autoimmune Encephalomyelitis (EAE) studies. The protocol involves bacterial lysis, sonication, centrifugation, and nickel affinity chromatography to obtain purified MOGtag. This protein enables B-cell-mediated autoimmunity research in multiple sclerosis models.

Protocol

1. 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.

1. Take one 1 ml aliquot from one of the overnight cultures and transfer it into a 1.5 ml microcentrifuge tube. Pellet the cells at 16,000 x g for 1 min and remove the supernatant. Label the tube TO/N (post-induction), then put the bacterial pellet into a -20 °C freezer for future SDS pag....

Results

Figure 1: Overview of the steps required to produce pure MOGtag protein.

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.
0.5M 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
Ethylenediamine tetraacetic acid, disodium salt dihydrate	Fisher scientific	BP120-500
Polyethyleneglycol 3350	Bioshop	PEG335.1
polyethyleneglycol 8000	Bioshop	PEG800.1
Sonicator	Fisher scientific	FB-120-110
Sodium dodecyl sulphate	Bioshop	SDS001