Isolation and Purification of Recombinant Myelin Oligodendrocyte Glycoproteins

Samenvatting

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 page analysis (Figure 2).
2. Distribute the cultures evenly amongst 250 ml bottles compatible with high speed centrifugation and keep these on ice from this point forward. Pellet the bacterial cells at 22,000 x g for 15 min at 4 °C.
3. Discard the supernatant. Store the bacterial pellets at -20 °C or continue to step 1.4.
4. Prepare lysis buffer (0.1 mg/ml hen egg lysozyme, 0.1% Triton-X (v/v) in phosphate-buffered saline (PBS)) by adding 60 µl of Triton X-100 and 120 µl of 50 mg/ml hen egg lysozyme stock solution to 60 mL of PBS.
5. Resuspend and combine all of the bacterial pellets from step 1.3 in a total of 30 ml of lysis buffer. Transfer this volume to a round-bottom 50 ml tube capable of high-speed centrifugation. Place this tube in a 30 °C water bath for 30 min. During the incubation time, shake the tube twice to resuspend the cells.
6. After the incubation, transfer the tube onto the ice and sonicate the solution at 20 kHz, amplitude 70%, pulse on for 3 sec, pulse off for 3 sec, and five pulses per round. Sonicate the solution for six total rounds and allow the solution to cool on ice in-between rounds.
7. Centrifuge the solution at 24,000 x g for 15 min at 4 °C. Discard the supernatant and repeat steps 1.5-1.7 once. Store the pellet at -20 °C or continue to step 1.8.
8. Prepare buffer A (500 mM NaCl (sodium chloride), 20 mM Tris-HCl (Tris (hydroxymethyl)aminomethane hydrochloride), 5 mM imidazole, pH 7.9) by adding 0.8766 g of NaCl, 0.09456 g of Tris-HCl, and 0.01021 g of imidazole to a 100 ml beaker. Add water until the volume reaches 28 ml, then adjust the pH of the solution to 7.9. Then, transfer the solution to a 100 ml graduated cylinder and add water until the volume is 30 ml.
9. Resuspend the pellet from step 1.7 in 30 ml of buffer A and incubate this solution at 4 °C for 3 hr. During this time, weigh out 17.2 g of guanidine-HCl.
10. Sonicate the solution on ice using the same settings as in step 1.6. Then add 17.2 g of guanidine-HCl to the solution. Incubate this on ice for 1 hr to solubilize the MOGtag protein.
11. Centrifuge the solution at 24,000 x g for 30 min at 4 °C. Collect the supernatant and store the supernatant at 4 °C until protein purification.

2. 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.

1. Make the Following Buffers:
   1. Prepare Buffer B (500 mM NaCl, 20 mM Tris-HCl, 5 mM imidazole, 6 M guanidine-HCl, pH 7.9). Add 5.844 g of NaCl, 0.6304 g of Tris-HCl, 0.0681 g of imidazole, and 114.64 g of guanidine-HCl to a 500 ml beaker. Then add water until it reaches 190 ml and adjust the pH to 7.9. Transfer the solution to a 250 ml graduated cylinder and add water until the volume is 200 ml.
   2. Prepare Elution buffer (500 mM NaCl, 20 mM Tris-HCl, 0.5 M imidazole, 6 M guanidine-HCl, pH 7.9). Add 5.844 g of NaCl, 0.6304 g of Tris-HCl, 6.808 g of imidazole, and 114.64 g of guanidine-HCl to a 500 mL beaker. Add water until it reaches 190 ml and adjust the pH to 7.9. Transfer the solution to a 250 ml graduated cylinder and add water until the volume is 200 ml.
   3. Prepare Strip buffer (500 mM NaCl, 20 mM Tris-HCl, 100 mM EDTA (ethylene diamine tetraacetic acid), pH 7.9). Add 5.844 g of NaCl, 0.6304 g of Tris-HCl, 40 ml 500 mM EDTA to a 500 mL beaker. Add water until it reaches 190 mL and adjust the pH to 7.9. Transfer the solution to a 250 ml graduated cylinder and add water until the volume is 200 ml.
   4. Prepare Charge buffer (0.1 M nickel sulfate). Add 5.257 g nickel sulfate to a 500 ml beaker and add water until it reaches 190 ml (CAUTION, do not handle nickel sulfate outside of a fume hood until the nickel sulfate has been dissolved in water). Once the nickel sulfate has dissolved, transfer the solution to a 250 ml graduated cylinder and add water until the volume reaches 200 ml.
2. Split 10 ml of nickel resin between two conical 50 ml centrifuge tubes capable of withstanding centrifugal forces over 4,500 x g (5 ml in each tube).
3. Charge and equilibrate the nickel resin:
   1. Wash the resin by adding 40 ml of water to each tube containing the resin. Lay the tubes horizontally onto a rocker and let them agitate for 5 min at 4 °C. Once finished, centrifuge the tubes at 4,500 x g for 8 min at 4 °C.
   2. Discard the supernatant by pipetting to avoid disturbing the pellet. Add 40 ml of charge buffer to each tube. Transfer the tubes onto a rocker and let them agitate for 15 min at 4 °C. Once finished, centrifuge the tubes at 4,500 x g for 8 min at 4 °C.
   3. Discard the supernatant, then add 40 ml of buffer B to the tubes. Transfer the tubes onto a rocker and let them agitate for 5 min at 4 °C. Once finished, centrifuge the tubes at 4,500 x g for 8 min at 4 °C, then discard the supernatants.
4. Optional: Take 150 µl of the solubilized protein collected in step 1.11 and transfer it to a 1.5 ml microcentrifuge tube. Label the tube as pre-incubation and freeze this at -20 °C for future SDS (sodium dodecyl sulfate) page analysis (Figure 2, Crude MOGtag).
5. Purify the MOGtag Protein:
   1. Transfer the entire volume of solubilized protein from step 1.11 (~40 ml, minus the small sample removed in step 2.4) to the first tube (Figure 1, tube 1) containing nickel resin from step 2.3, mix, and place horizontally on a rocker at 4 °C for 1 hr.
   2. Centrifuge the tube at 4,500 x g for 8 min at 4 °C. Once finished, transfer the supernatant to the second tube (Figure 1, tube 2) of nickel resin and incubate as described in the previous step. In the meantime, continue with steps 3 to 6 below with tube 1.
   3. Resuspend the nickel resin in tube 1 in 40 ml of elution buffer and place the tube horizontally on a rocker at 4 °C for 5 min. Then, centrifuge the tube at 4,500 x g for 8 min at 4 °C.
   4. Transfer the supernatant containing eluted MOGtag protein into a 250 ml bottle labelled 'purified MOGtag protein' and keep this bottle at 4 °C. With each elution step, pool the resulting supernatant in this bottle.
   5. Add 40 ml of strip buffer to the nickel resin and place horizontally on a rocker for 5 min at 4 °C.
   6. Centrifuge the tube at 4,500 x g for 8 min at 4 °C. Discard the supernatant, then recharge the nickel resin as listed in step 2.3.
   7. Once finished, move forward with the second tube as listed in step 2.5. A total of 4 rounds of absorption of the solubilized protein from step 1.11 onto the charged nickel resin and elution will recover most of the protein although, if desired, additional protein could be recovered in additional rounds of absorption and elution.
6. Once four (or more) rounds of absorption and elution are complete, store the pooled purified protein at 4 °C overnight. Nickel resin can be stored in 40 ml of a 20% ethanol solution at 4 °C until it is needed again.

Resultaten

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

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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