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Preparation of dolichylpyrophosphoryl oligosaccharides from Saccharomyces cerevisiae
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Preparation of dolichylpyrophosphoryl oligosaccharides from Saccharomyces cerevisiae

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Introduction Protocol References Credit lines
Category
Biosynthesis & Metabolism
Protocol Name

Preparation of dolichylpyrophosphoryl oligosaccharides from Saccharomyces cerevisiae

Authors
Harada, Yoichiro
Glycometabolome Team, RIKEN Advanced Science Institute
KeyWords
Reagents

Yeast cells (In this protocol, BY4741 (purchased from Thermo Fisher Scientific Inc., Waltham, MA) haploid cells were used.)

YPD medium for yeast culture, autoclaved (YPD: yeast extract, 10 g/L; tryptone, 20 g/L and glucose, 20 g/L)

Phosphate-buffered saline (PBS)

Lysis buffer (20 mM Hepes-KOH, pH 7.4, 2 mM EDTA, 200 mM sorbitol, 50 mM potassium acetate)

Resuspension buffer (20 mM Tris-HCl, pH 7.4 and 250 mM sucrose)

1 M MgCl2 in water

Water (abbreviated as W)

Methanol (abbreviated as M)

Chloroform (abbreviated as C)

Isopropanol

Graphitized carbon column (150 mg/3 mL, GL Sciences Inc., Tokyo, Japan)

Dowex AG1-X2 (200–400 mesh, acetate form) (Bio-Rad Laboratories, Hercules, CA)

Dowex AG50-X8 (200–400 mesh, H+ form) (Bio-Rad Laboratories)

Polyprep column (10 mL, Bio-Rad Laboratories)

Instruments

Shaker for yeast culture (Excella E24, New Brunswick Scientific Co., Inc. Edison NJ)

Glass beads (0.5 mm, Yasui Kikai Corp., Osaka, Japan)

Gauze or equivalent

Funnel

Glass tube with screw cap (PYREX)

Bead-beater (Biospec Products Inc., Bartlesville, OK)

Centrifuge (Avanti HP-30I, Beckman Coulter, Inc., Brea, CA) and (himac CF8DL, Hitachi, Ltd., Tokyo, Japan)

Ultracentrifuge (CP-80WX, Hitachi Ltd.)

Probe-type sonicator (or water bath-type sonicator) (VC750, Sonics & Materials, Inc., Newtown, CT)

Incubator that can heat up to over 100ºC (NDS-400, EYELA, Tokyo, Japan)

Speed Vac (Centrivap 78100, Labconco Corp., Kansas City, MO)

Methods
1.

Yeast culture

1) 

 Pre-culture yeast cells in YPD medium at 30ºC for 18 h.

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2) 

 Prepare six 2-L flasks containing 1 L each of YPD.

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3) 

 Add 1 mL of the pre-culture to each flask.

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4) 

 Incubate the culture at 200 rpm at 30ºC until an optical density at 600 nm (OD600) reaches 7–10. This incubation step may take 16 h.

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2.

Preparation of the microsomes from yeast

1) 

 Harvest the cells by centrifugation at 3,000 rpm for 5 min at 4ºC and discard the supernatant.

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2) 

 Resuspend the cells with 100 mL of water and centrifuge at 3,000 rpm for 5 min at 4ºC.

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3) 

 Discard the supernatant.

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4) 

 Repeat 2-2) and 2-3) once.

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5) 

 Resuspend the cells with 100 mL of PBS and centrifuge at 3,000 rpm for 5 min at 4ºC.

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6) 

 Discard the supernatant.

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7) 

 Resuspend the cell with Lysis buffer and adjust the volume to approximately 150 mL.

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8) 

 Transfer the cell suspension to a bead-beater chamber.

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9) 

 Add approximately 150 mL of ice-cold glass beads.

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10) 

 Lyse the cell by sixteen 10-sec homogenizations separated by 10-sec cooling periods on ice.

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11) 

 Filtrate the homogenate into a glass beaker through a funnel fitted with gauzes.

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12) 

 Centrifuge the filtrate at 3,000 rpm for 5 min at 4ºC and recover the supernatant.

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13) 

 Centrifuge the supernatant at 6,000 × g for 5 min at 4ºC and recover the supernatant.

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14) 

 Ultracentrifuge the supernatant at 100,000 × g for 1 h at 4ºC and discard the supernatant.

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15) 

 Resuspend the pellet (microsomes) with a small volume of resuspension buffer.

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16) 

 The final volume of the microsomes may be 5–10 mL.

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17) 

 If the microsomes become jelly-like substances, pass the microsomes 10–20 times through 18G and 22G needles sequentially.

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18) 

 The microsomes may be flash frozen in liquid nitrogen and stored at −80ºC until use (Not recommended though).

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3.

Extraction of Dol-PP-OS from the microsomes

1) 

 If the microsomes are frozen, thaw on ice slowly.

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2) 

 Transfer 2 mL of the microsomes into a glass tube with screw cap.

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3) 

 Add 30 mL of ice-cold C/M (2:1, v/v), vortex and sonicate until any aggregate becomes dispersed.

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4) 

 Centrifuge at 3,000 rpm for 10 min at 4ºC and discard the supernatant.

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5) 

 Repeat 3-3) and 3-4) once.

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6) 

 Dry the pellet under nitrogen stream.

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7) 

 Add 30 mL of ice-cold M/W (1:1, v/v) containing 4 mM MgCl2, vortex and sonicate until any aggregate becomes dispersed.

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8) 

 Centrifuge at 3,000 rpm for 10 min at 4ºC and discard the supernatant.

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9) 

 Repeat 3-7) and 3-8) once.

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10) 

 Dry the pellet under nitrogen stream.

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11) 

 Add 4 mL of C/M/W (10:10:3, v/v/v, room temperature; the temperature is IMPORTANT!), vortex, sonicate until any aggregate becomes dispersed and then incubate at 37ºC for 10 min.

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12) 

 Centrifuge at 3,000 rpm for 10 min at room temperature (IMPORTANT!) and save the supernatant.

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13) 

 Repeat 3-11) and 3-12) once and save the supernatant.

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14) 

 Combine the first and second supernatants and dry in vacuo.

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15) 

 Extract Dol-PP-OS from the dried pellet by addition of 4 mL of C/M/W (10:10:3, v/v/v, room temperature; the temperature is IMPORTANT!) and vortex.

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16) 

 Centrifuge at 3,000 rpm for 10 min at room temperature and save the supernatant.

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17) 

 Repeat 3-15) and 3-16) once and save the supernatant.

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18) 

 Combine the first and second supernatant and dry in vacuo.

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19) 

 Dissolve the pellet in a small volume (~ 1 mL) of C/M/W (10:10:3, v/v/v, room temperature). The resultant solution contains the partially purified Dol-PP-OS.

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20) 

 Store the partially purified Dol-PP-OS at −80ºC.

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4.

Release of glycan from the Dol-PP-OS preparation by mild acid hydrolysis (Optional)

1) 

 Transfer various volumes of the Dol-PP-OS preparation to glass tubes with screw cap.

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2) 

 Dry in vacuo.

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3) 

 Add 500 μL of isopropanol and vortex.

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4) 

 Add 500 μL of 40 mM HCl and vortex (proceed the next step even you see some aggregates).

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5) 

 Heat the glass tubes at 100ºC for 30 min.

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6) 

 Cool the tubes in a water bath.

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7) 

 Dry in vacuo.

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8) 

 Add 1 mL of water, vortex and centrifuge at 3,000 rpm for 10 min at 4ºC.

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9) 

 Save the supernatant (it contains the released glycans).

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10) 

 Repeat 4-8) and 4-9) twice.

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11) 

 Apply the combined supernatant (approximately 3 mL) to a graphitized carbon column.

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12) 

 Wash the column with 3 mL of water.

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13) 

 Elute the glycan with 2.5 mL of 25% acetonitrile in water.

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14) 

 Dry the eluate in vacuo.

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15) 

 Dissolve the dried samples in water and apply onto a coupled ion exchange chromatography (250 μL each of Dowex AG1-X2 and Dowex AG50-X8, resins should be carefully packed in a Polyprep column).

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16) 

 Collect the flow-through.

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17) 

 Wash the column with 1.5 mL of water and save the flow-through.

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18) 

 Combine all the flow-through fractions and dry in vacuo.

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19) 

 Label the glycan with 2-aminopyridine and analyze them by high performance liquid chromatography (HPLC) as described elsewhere. The glycans were quantitated by using PA-isomaltohexaose included in PA-glucose oligomer DP=3-15 (Takara Bio Inc., Otsu, Japan) as a standard.

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Initial amount

Total OD600 is 54,000.

Produced amount

Approximately 10 nmol of total DLOs as PA-isomaltohexose.

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