TITLE: Purification of Methylglucose Lipopolysaccharides (MGLP)
AUTHORS: Briana Marisco, Luisa M Nieto Ramirez, Karen Dobos

[DESCRIPTION]
Methylglucose lipopolysaccharides (mGLPs) are poly-methylated polysaccharides (PMPSs) that are acylated with short-chain fatty acids, uniquely synthesized by bacteria belonging to the Actinomycetales order. mGLPs are composed of ~20 glucose (Glc) units, most of them O-methylated (6-O-Me-Glc, Glc, and a 3-O-Me-Glc in a ratio of 11:8:1). Acyl functions in mGLPs are heterogeneous and contain acetyl, isobutyryl, succinyl, and octanoyl groups. mGLPs also known as 6-O-methylglucose-containing-lipopolysaccharides are believed to be essential in Mycobacterium tuberculosis and participate in the regulation of lipid metabolism, protecting fatty acids from degradation.  Additionally, this cytosolic bacterial component is a potent stimulator of γ9δ2 T cells.




[STEPS]
SECTION: Total lipid extraction
1. Obtain approximately 100 g   Mycobacterium tuberculosis (Mtb) cells and freeze dry by lyophilization.

 

SECTION: Total lipid extraction
2. Determine the dry weight of the cells and transfer them to a 500 mL glass bottle.
SECTION: Total lipid extraction
3. Delipidate cells by adding 10mL of 10:10:3 [vol/vol/vol] chloroform: methanol: water per 1 gr of the Mtb cells. Add a stir bar and stir   in a chemical fume hood. Make sure the bottle is tightly closed.

SECTION: Total lipid extraction
4. Transfer overnight extract into 35 mL Teflon Oak Ridge tube(s) and centrifuge at 27000 x g, 20 min, 15 °C 
Note: The number of Teflon tubes would depend on the volume obtained in step 3.

SECTION: Total lipid extraction
5. Decant organic supernatant and preserve it in a labeled bottle.

SECTION: Total lipid extraction
6. Transfer the cells from tubes back to 500 mL bottle and repeat delipidation (steps 2 -5) two more times, reducing incubation time to 2 hours for each repeat. Be sure to vortex the cell pellet for one minute before repeating. 

 


SECTION: Total lipid extraction
7. Collect all extracts into the same bottle (started on step 5).
SECTION: Total lipid extraction
8. Measure total volume and aliquot 0.5 mL into a previously weighed 2 mL glass vial. Dry under a Nitrogen bath and calculate mg/mL of total volume based on the net weight obtained.
SECTION: Removal of high molecular weight molecules
10. Prepare your Amicon‱ Ultra-15 Centrifugal Filter Unit 10 kDa MWCO by adding 5 mL of MiliQ water and centrifuging at 3500 x g, 5 min, 20 °C. Dispose the flow-through. 
 


SECTION: Removal of high molecular weight molecules
11. Add your total lipid extract from step 9 to the Amicon‱ Ultra-15 Centrifugal Filter Unit 10 kDa MWCO and centrifuge at  3500 x g, 30 min, 20 °C, keeping the flow-through. Check volume level: if there is only a small amount of flow-through, increase speed to 3750 – 4000 x g.

SECTION: Removal of high molecular weight molecules
12. Repeat centrifugation until most of the volume is passed  and the retentate volume is ~ 1 mL.
SECTION: Removal of high molecular weight molecules
13. Rinse 10 kDa filter membrane using Pasteur pipette by adding 2 – 3 mL of MiliQ water, pipette up and down several times. Centrifuge for 10 minutes and collect the flow-through.
 

16. Transfer retentate from previous step to 13 x 100 tube and thoroughly rinse 3 kDa filter 2X with 1 mL of water, pipetting up and down to collect any residual product before transferring to 13 x 100 tube. Record the total volume obtained. 
17. Determine the total amount of crude material in 3 kDa retentate by aliquoting 0.5 mL into a pre-weighted 2 mL vial. Calculate mg/mL of total 3KDa crude material.
18. Aliquot 0.5 mg of 3 kDa retentate into a 13 x 100 tube and completely dry down on a N2 bath. Resuspend in 500 uL of water and dilute 1:10 by adding 4.5 mL of additional water. 
 

19. Prepare your Sep-Pak C18 cartridge, adding in order the following solutions, using a 10 mL glass syringe:
a. 4 mL 95% acetonitrile 
b. 5 mL 100% methanol
c. 2 mL 80% methanol in chloroform
20. Equilibrate your cartridge by adding 3 mL of water; leave enough water in the column so that it is saturated and a droplet is visible at the bottom. Let sit for 5 minutes.
21. Load your diluted crude sample into the column and slowly elute.
22. Wash with 4 mL of water (no need to collect the flow-through).
23. Perform a series of elutions with the following, with a wash step in between each elution, using 4 mL of water :
a. 4 mL 30% methanol in water
b. 4 mL 60% methanol in water
c. 4 mL 90% methanol in water

Collect each elution and washes steps in a labeled and pre-weighted 13 x 100 tube.

 

26. Determine the dried weight of the 60% and 90% fraction and resuspend in 0.5 mL of water. Record the concentration obtained for each fraction. 
24. Repeat Sep-Pak chromatography for as much material is needed. Around 6 to 8 cycles of Sep-Pak preparations would be needed for 50 mg of the 10:10:3 extract (obtained in step 9).  

29. Perform the  MALDI-TOF for QC. 
 

 

SECTION: Total lipid extraction
9. Take 50 mg of your 10:10:3 total lipid and completely dry down via  Nitrogen bath. Resuspend in MilliQ water at a concentration of 5 mg/mL (10 mL total). Use a water bath sonicator to thoroughly resuspend – may take several minutes. If necessary, can also add more water to further dilute and ensure complete resuspension.

 


SECTION: Concentration of mGLP (~3.5-4 kDa)
14. Transfer pooled flow-through (from steps 11-13) to a Cytiva Microsep Advance Centrifugal Devices with Omega Membrane 3kDa (maximum capacity 4 mL). 

SECTION: Concentration of mGLP (~3.5-4 kDa)
15. Centrifuge at 3500 x g, 20 °C  for 10 minutes and dispose of flow-through. Add more of the 10 kDa flow-through until max capacity (4 mL) is reached again in the Cytiva Microsep. 

Continue this process until all the 10 kDa flow-through has been passed through the 3 kDa filter and the retentate volume is reduced to ~ 1 mL.
25. In the N2 bath, dry the 60%, and 90% fractions obtained in the Sep-Pak runs, separately (mGLP is enriched mostly in the 60% fraction and in a less extent in the 90% fraction).
27. Apply 10μg -20μg of these fractions into an analytical TLC plate and run the plate in 56:38:10 Chloroform: Methanol: Water. After this, stain the TLC plate with α-Naphtol.


 

28. After combining the 60% and 90% fractions, the yield of purified mGLP is about 4% of the 10:10:3 obtained material from step 9 (starting with 50 mg of the 10:10:3 extract, the yield of is approximately 4 mg).