Team:Washington/Protocols/1LPurificationCapD

Protein Expression/Purification of Recombinant Protein in pET29b+

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 50mL LB starter cultures in STERILE 250mL flask:
  • a.Thoroughly wash Flask with HOT WATER
    • i.Removes trace bleach/soap/etc which can inhibit cell growth
  • b.RINSE WITH diH2O (remove tap water particulates)
  • c.Autoclave for 20-40min
  • d.Cool down (1hr bench top should be enough)
  • e.50mL LB, 50uL 50mg/mL KAN
  • f.Innoculate with 1 colony or a scrape from glycerol stock
  • g.Grow O/N at 37C with shaking at 250 (let grow for >=18hrs).
  • h.Make glycerol stock using 500uL 50% glycerol + 500uL O/N culture.
    • i.This is only necessary if you do not already have a glycerol stock
• 3.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.
    • i.Autoclave
    • ii.Let Cool down
    • iii.Make AUTOMIX (In separate 50ml falcon tube):
      • 1.500uL 1M MgSO4
      • 2.500uL 1000X Metals Mix
      • 3.25mL 20X NPS
      • 4.10mL 50X 5052
      • 5.500uL 50mg/mL KAN
    • iv.Add contents of AUTMIX tube to Media
    • v.Add remaining 49.5mL of starter culture to media
    • vi.Grow at 18C for 24-30 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 500mM, 25mM Imidazole (pH 8)
  • b.Lysis Buffer: 50mM HEPES (pH 8), NaCl 500mM, 25mM Imidazole (pH 8), 2x Bug Buster, 2mg/mL lysozyme, 0.2mg/mL DNAse
  • c.Elution buffer: 50mM HEPES (pH 8), NaCl 500mM, 500mM Imidazole (pH 8)
  • d.Talon strip buffer: 100mM EDTA, pH 8.0
  • e.Talon recharge buffer: 100mM CoCL2
• 2.Lyse Cells (Sonication)
  • a.Sonication
    • i.Make master mix of lysis buffer and add 10mL to each pellet and resuspend. Vortexing ok.
• 3.After each pellet is resuspended, pour resuspension into cleaned SS34 tube (holds 60mL roughly).
• 4.Add more lysis buffer so that its roughly 4/5 full
• 5.Remove Insoluble Matter
  • a.Balance samples in SS34 tubes (pairwise is sufficient)
  • b.Spin at >=18000rpm for >=30min at 4-15deg
    • i.If supernatent is still “goopy” add a little more DNAse and spin longer
• 6.Remove Particles
  • a.Filter supernatant with 0.4uM Filter into new 50mL falcon tube.
    • i.If analyzing expression save Supernatant & Pellet Gel Samples (50uL)
• 7.Run Proteins over Column
  • a.Equilibrate 1mL NiNTA-Superflow Gravity columns with 10mL wash buffer.
  • b.Run supernatant over column 2X
    • i.If analyzing expression Save Load FT. Load FT Gel Sample
  • c.Wash column 1x with 20mL wash buffer with .5% Tween.
    • i.If analyzing expression Save Wash FT Gel Sample
  • d.Wash column 1x with 20mL wash buffer no Tween
  • e.Elute column with 15mL elution buffer
    • i.Collect in Vivaspin-20 Concentrator.
  • f.Save excess Elution buffer for blanking/diluting proteins later
• 8.Concentrate Proteins
  • a.Check A280 on using spec (e.g. nanodrop)
  • b.Spin at 8000 rpm for about 10-20min.
  • c.Target volume is 200uL
    • i.If this puts A280 above 30 stop at a larger volume, proteins can crash out when too concentrated.
• 7.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 1-4L of Dialysis buffer for at least 2 hours (overnight is fine)
  • c.Repeat so at least two rounds of dialysis have been done
• 8.Determine Protein Concentration
  • a.Check A280
  • 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
• 9.Store protein
  • a.For short term store in 4deg
  • b.For long term flash freeze and store at -80deg
• 10.Clean columns
  • a.10mL 5M GuHCl + 500mM Imidizole + 1% nonionic detergent (Tween-20)
  • b.10mL of ddiH2O
  • c.10mL of 0.2M EDTA (pH ~7.0)
  • d.10mL of ddiH2O
  • e.10mL of 50mM CoCl2
  • f.10mL of ddiH2O
  • g.10mL of 300mM NaCl
  • h.10mL of ddiH2O
  • i.10mL of 20% EtOH
  • j.Cap bottom, add 5mL of 20% EtOH, cap top and store

Buffers

Wash Buffer (GENERAL STOCK BUFFER):

• 50mM pH 7.4 HEPES
• 500mM NaCl
• 25mM Imidizole

Lysis Buffer:

• 50mM pH 7.4 HEPES
• 500mM NaCl
• 25mM Imidizole
• 2mg/ml lysozyme (small scoop)
• 0.2mg/ml DNAse (small scoop)

Elution Buffer:

• 50mM pH 7.4 HEPES
• 500mM NaCl
• 200mM Imidizole