Matsudaira:hsCen2 purification

Danielle France October 2005

Purification of human centrin 2 from E. coli

vector: pGEX-6P
contains lacIq gene (repressor), tac promoter, Ampr gene (resistance to 100 μg/mL ampicillin), hsCen2 gene [hsCen2 ~ 20 kDa], GST gene = fusion protein used as tag for purification [GST ~ 26 kDa], PreScission™ Protease cleavage site between hsCen2 & GST

• protocol closely follows that given by Amersham in pGEX system handbook

Wet Materials

for Growth & Induction

• 2x YTA (1L)
  • 16g tryptone
  • 10g yeast extract
  • 5g NaCl
    1. Dissolve above in 900 mL milliQ H2O.
    2. Adjust pH to 7.0 with NaOH. Adjust volume to 1000 mL.
    3. Sterilize by autoclaving for 20 min.
  • 100 µg/mL ampicillin
    1. Let cool to 50oC, then aseptically add 1 mL of a 100 mg/mL ampicillin stock solution.

• 100 mM IPTG
• ice-cold PBS
  

for Lysis & Solubilization

• Protease inhibitors
  • aprotinin (stored at 4oC)
  • leupeptin (stored at –20oC in dmso, can thaw/refreeze)
  • IMBA / benzamidine (stored at –20oC, do not thaw/refreeze)
    

use each at 1:1000 dilution of stock

• 1x PBS (ice-cold)
• 20% Triton X-100
• 10x SDS gel loading buffer

for Batch Purification

• Glutathione Sepharose 4 Fast Flow

Amersham product #17-5132-01

• Elution buffer (15 mL): Make just before using.
  • 750 μL 1M Tris-HCl
  • 14.25 mL milliQ water
  • .0461 g reduced glutathione
    1. Dissolve reduced glutathione into buffer check for pH~8.0. If elution appears incomplete, amount of glutathione can be raised.

(50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0)

Procedure

→Be sure to keep track of all times, volumes, and absorbance or concentration readings for trouble-shooting purposes.

Growth and induction

Ref: Amersham GST protocols, pp 23-24, “9. Preparation of large-scale bacterial sonicates.”

1. Use a single colony of E. coli to inoculate 2-100 mL of 2x YTA medium
2. Incubate for 12-15 hrs at 37oC with vigorous shaking.
   • Small volumes (<3mL): use rotator in tall grey incubator, room 654
   • Large volumes go into Lindquist 37oC room 674.
3. Dilute the culture 1:100 into fresh pre-warmed 2x YTA medium. Use 4L plastic flasks, with <1L medium per flask. Grow at 37oC on room 674 shaker until the A600 reaches 0.5-2 (generally about 2 hours). Be sure to balance flasks on shaker platform.
   • A600,pre-growth =
4. Add 100 mM IPTG to a final concentration of 0.33 mM and continue incubation for an additional 4 hrs.
   • A600,pre-induction =
5. Transfer the culture to 1L flat-bottomed centrifuge bottles and spin at 5000 RPM in large 6-bucket centrifuge for 35 min at 4oC. A600, post-induction
6. Siphon off supernatant and resuspend pellet (using small volume of cold PBS if necessary). Combine pellets into one 250 mL or 500 mL conical centrifuge bottle.
7. Spin at 5000 RPM in large 6-bucket centrifuge for 15 min at 4oC.
   

Continue to cell lysis immediately or freeze pellet at -20oC.

Lysis and solubilization

For all samples, take 90 µL of material and mix with 10 µL SDS sample loading buffer. Boil 5-10 min at 95oC (in heat block on Guichy’s bench), then freeze immediately in dry ice and store at -20oC.

1. Resuspend pellet in 50 μL of ice-cold PBS per mL of original culture volume.
   • take sample 0: resuspended cells
2. Distribute the suspended cells into plastic 50 mL tubes. Disrupt cells using the Lindquist Lab sonicator (room 606A, entered through room 688). Sonicate on ice at power level 10 for 30 sec, then rest 30 sec. Repeat twice for a total of three 30-sec bursts.
3. Immediately add protease inhibitors: leupeptin, aprotinin, pepstatin, at 1:1000 dilution.
4. Add 20% Triton X-100 to a final concentration of 1%. Rotate gently in cold room for 30 min to aid in solubilization of GST-hsCen2.
5. make sure heat block on Guichy’s bench is on at 95oC
6. Centrifuge at ~12000g (10k RPM on Sorvall-SS-34) for 15 min at 4oC.
7. Transfer S/N to fresh container. Take sample.
   • take sample 1s/n: supernatant containing soluble proteins, including hsCen2.
8. Wash pellet with small amount of distilled H2O and resuspend in same volume of PBS as supernatant. Take sample and discard remaining pellet.
   • take sample 1p: pellet containing insoluble proteins & other cellular junk

Continue to purification immediately or freeze supernatant at -20oC.

Batch purification on Glutathione Sepharose 4 Fast Flow

Ref: Amersham GST protocols, pp 38-40, “13.1. Purification using Glutathione Sepharose 4B medium.”

1. Prepare medium. It comes as a 60% slurry in 20% ethanol. For every 100 mL of your cell lysate S/N, you will need 5 mL of 50% slurry in PBS. Calculate the volume of 60% slurry you will need to start.
   1. Gently shake the bottle of Glutathione Sepharose 4 FF to resuspend the medium.
   2. Use a pipet with a wide-bore tip to remove sufficient slurry, and transfer slurry to a (250 mL) centrifuge tube.
   3. Sediment the medium by centrifuging at 500g for 8 min. (1.8k RPM on 6-bucket floor centrifuge). Drain off supernatant.
   4. Resuspend medium in 10 mL of cold PBS for every 1.33 mL of original 60% slurry used. Invert to mix.
   5. Sediment the medium by centrifuging at 500g for 8 min. (1.8k RPM on 6-bucket floor centrifuge). Drain off supernatant.
   6. For each 1.1 ml of the original slurry dispensed, add 1 mL of PBS. This results in ~ 50% slurry. Mix well prior to subsequent steps.
   • bed volume = ½ of 50% slurry volume
2. Start batch purification. Add 100 mL of your cell lysate S/N for each 5 mL of the 50% slurry of Glutathione Sepharose 4 FF to the (250 mL) centrifuge tube.
3. Incubate for 60 min at 4oC. Use gentle agitation such as end-over-end rotation or tilt table.
4. Sediment the medium by centrifuging at 500g for 8 min. (1.8k RPM on 6-bucket floor centrifuge). Carefully pour off S/N; save in separate tube and label as 1st flow-through.
   • Take sample 2 Vo: proteins which did not bind to GS4FF medium
5. Resuspend in 10 bed volumes of PBS. Invert to mix.
6. Sediment the medium by centrifuging at 500g for 8 min. (1.8k RPM on 6-bucket floor centrifuge). Carefully pour off S/N; save in separate tube and label as 1st wash. #*Take sample 3a s/n:wash of medium to remove more unbound proteins
7. Repeat steps 5 and 6 twice for a total of three washes. Continue to save, label, and sample all supernatants.
   • Take sample 3b s/n
   • Take sample 3c s/n
8. Add 1.0 mL of elution buffer per 1 mL bed volume of the original slurry. Mix gently to resuspend.
9. Incubate for 20 min at 4oC to elute GST-hsCen2 from the medium. Use gentle agitation such as end-over-end rotation or tilt table.
10. Sediment the medium by centrifuging at 500g for 8 min. (1.8k RPM on 6-bucket floor centrifuge). Carefully remove S/N; in contains your GST-hsCen2! Save in separate tube and label as “GST-hsCen2 elution 1”.
    • Take sample 4a s/n: eluted protein
11. Repeat steps 8-10 twice for a total of three elutions.
    • Take sample 4b s/n
    • Take sample 4c s/n

If needed, continue to more chromatography immediately or freeze all collected samples on dry ice and store at -20oC.

• Run SDS gel to analyze content of all samples and determine results of purification.

**10 samples + MW standards: needs 15 lane gel