From 2012.igem.org

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

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.

Team:Washington/Protocols/expression purification

From 2012.igem.org

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

Contents

Expression

Purification

Buffers