From 2011.igem.org

Latest revision as of 00:12, 29 September 2011

Protein Expression/Purification of Recombinant Protein in pET29b+ Using Autoinduction

Adopted from [http://www.sciencedirect.com/science/article/pii/S1046592805000264 Protein production by auto-induction in high-density shaking cultures by Studier]

Expression

• 1.Transform pET29b+ DNA into BL21(DE3)* competent cells, plate on KAN.
  • a.You can skip this step if there is already a glycerol stock in BL21(DE3)*
  • b.Combine 1uL of DNA + 20uL of cells in Falcon tube
    • i.Eppendorf is fine as well
  • c.Quickly immerse tube with cells in water batch at 42deg for 45sec
  • d.Remove and let rest on ice for 2min
  • e.Add 200uL of LB
  • f.Let recover, shaking at 37deg for 45min
  • g.Plate 200uL of cells onto a LB/Kan plate
• 2.Grow 0.5L cultures, in ZY Auto Induction media in 2L flask:
  • a.Thoroughly rinse out flask with HOT WATER
    • i.Removes trace bleach/soap/etc which can inhibit cell growth
  • b.RINSE WITH diH2O (remove tap water particulates)
  • c.5g Tryptone, 2.5g Yeast Extract + 465mL H2O. <---PREP THIS THE DAY BEFORE
    • i.Autoclave
    • ii.Let Cool down overnight
    • iii.Add Autoinduction Components
      • 500uL 1M MgSO4
      • 500uL 1000X Metals Mix
      • 25mL 20X NPS
      • 10mL 50X 5052
      • 500uL 50mg/mL KAN
    • iv.Add cells to innoculate (100uL of an overnight culture or a scrape from a plate)
    • v.Grow at 37C for 3-6 hours (until visibly cloudy)
    • vi.Turn temp down to 18C and continue growth for 20-30 hours.
• 4.Collect Cells
• 5.Spin at 4000rpm for 20 minutes.
• 6.Discard supernatant
• 7.Resuspend pellet in 5mL 1x PBS, pH 7.4
• 8.Transfer to 50mL falcon tubes.
  • a.Run gel to confirm expression. (optional)

Purification

• 1.Prepare buffers (recipes on next page)
  • a.Wash buffer: 50mM HEPES (pH 8), NaCl 100mM, 25mM Imidazole (pH 8), 10% glycerol, 1mM TCEP
  • b.Lysis Buffer: 50mM HEPES (pH 8), NaCl 100mM, 25mM Imidazole (pH 8), 10% glycerol, 1mM TCEP, 2mg/mL lysozyme, 0.2mg/mL DNAse, 1mM PMSF
  • c.Elution buffer: 50mM HEPES (pH 8), NaCl 100mM, 200mM Imidazole (pH 8), 10% glycerol, 1mM TCEP
  • d.Dialysis buffer: 50mM HEPES (pH 8), NaCl 100mM, 10% glycerol, 1mM TCEP
  • d.NiNTA strip buffer: 100mM EDTA, pH 8.0
  • e.NiNTA recharge buffer: 100mM NiSO4
• 2.Lyse Cells (Sonication) -- ~5minutes/sample
  • a.Sonication
    • i.Make master mix of lysis buffer and add 15mL to each pellet and resuspend. Vortexing ok.
• 3.After each pellet is resuspended, pour resuspension into cleaned SS34 tube.
  • a.Add more lysis buffer if necessary so that its roughly 3/4 full
• 5.Remove Insoluble Matter -- ~1hr (setup and walk away)
  • a.Balance samples in SS34 tubes (pairwise is sufficient)
  • b.Spin at >=18000rpm for >=30min at 4-15deg
• 6. Bind to NiNTA Beads -- 20 minutes
  • a. In the cold room, setup BioRad columns (Prepacked and with 1mL NiNTA) over a waste collection tray
  • b. Let the 20% EtOH drain, was with 5mL of water, then wash with 5mL of your wash buffer
  • c. Filter the supernatent of your protein using a 0.4 - 0.8micron filter
  • d. Pour your filtered supernatent over the columns to let your protein bind
• 7. Wash Off Unbound Protein -- 1hr (pour on and walk away)
  • a.Wash column 3x with ~10-20mL wash buffer
• 8. Elute off Bound Protein -- 20minutes
  • a.Setup Vivaspin-20 Concentrator under columns
  • b.Add 15mL of elution buffer and collect in concentrators
• 9.Concentrate Proteins -- 30minutes
  • a/Spin at 8000 rpm for about 10-20min.
  • b.Target volume is 500uL
• 10.Remove Excess Imidazole
  • a.Transfer 800uL to a dialysis tube (midi, 50-800uL), save the remaining protein
    • i.Novagen Cat No 71507-3
  • b.Let equilibrate in 1L of Dialysis buffer for at least 2 hours in the cold room (overnight is fine)
  • c.Repeat so at least two rounds of dialysis have been done
• 11.Determine Protein Concentration
  • a.Check A280 on the NanoDrop
  • b.Calculate Protein Concentration
    • i. Go to the [http://web.expasy.org/protparam/ Expasy ProtParam] website
      • a. [http://web.expasy.org/translate/ Translate] your DNA sequence to protein sequence if necessary
    • ii. Enter your sequence and compute paramters
    • iii. Record the extinction coefficient of your protein
    • iv. A = E* L * C ; Path Length (L) From the nandrop is 1 cm, A is your A280 value from the nanodrop, E is your extinction coefficient from above (in M-1cm-1), C is your unknown concentration.
      • v. Example: Protein A280 is 3.3 and extinction coefficient is 24535, so concentration is: 0.000135 M or 135 uM
  • b.Run Gel of Samples
    • i.Make 1:10 dilution of lysis, supernatent
    • ii.Dilute preconcentrated and final protein so A280 ~1.5
    • iii.Combine 5uL sample with 5uL 2x SDS Loading Buffer.
    • iv.Boil for 5min
    • v.Run 4-20% BioRad Gel for 35min at 200v
    • vi.Wash 3x with diH2O (rock 5min between each rinse)
    • vii.Stain using Thermo staining reagent
• 12.Store protein
  • a.For short term store in 4deg
  • b.For long term flash freeze and store at -80deg
• 13.Clean columns (start in parelled with step 9) -- 40 minutes
  • b.10mL of ddiH2O
  • c.10mL of 0.1M EDTA (pH ~7.0)
  • d.10mL of ddiH2O
  • e.10mL of 100mM NiSO4
  • h.10mL of ddiH2O
  • i.10mL of 20% EtOH
  • j.Cap bottom, add 5mL of 20% EtOH, cap top and store

Buffers

• 100mM EDTA, pH 8.0 (1L)
  • 37.2g Na2EDTA *pH to 8.0, bring up to 1L with ddH20
    • Wont go into solution fully until pH is raied

• 100mM NiSO4 (1L)
  • 26.3g NiSO4*6H20 *bring up to 1L with ddH20

• 1M Imidazole (1L)
  • 68.1g Imidazole *bring up to 1L with ddH20

• 1000x Trace Metals Mix - 100ml
  • Compound Amount
  • Water 36ml
  • 0.1M FeCl3-6H2O 50ml
  • 1M CaCl2 2ml
  • 1M MnCl2-4H2O 1ml
  • 1M ZnSO4-7H2O 1ml
  • 0.2M CoCl2-6H2O 1ml
  • 0.1M CuCl2-H2O 2ml
  • 0.2M NiCl2-6H2O 1ml
  • 0.1M Na2MoO4-2H2O 2ml
  • 0.1M Na2SeO3-5H20 2ml
  • 0.1M H3BO3 2ml

• 20x NPS - 1L
  • NPS = 100mM PO4, 25mM SO4, 50mM NH4, 10mM Na, 50mM K
  • Compound Amount
  • water 900ml
  • (NH4)2SO4 66g
  • KH2PO4 (monobasic) 136g
  • Na2HPO4 (dibasic) 142g

• 1M MgSO4 - 300ml
  • Compound Amount
  • water 300ml
  • MgSO4 74g
    • After dissolved, sterile filter

• 50x 5052 - 1L
  • • 5052 = 0.5% glycerol, 0.05% glucose, 0.2% alpha-lactose
  • Compound Amount
  • water 730ml
  • Glycerol 250g
  • Glucose 25g
  • a- lactose (D+) 100g
    • Can speed up dissolving by heating in the microwave.
  • Sterile Filter
  • Store in refrigerator.