Protocol for harvesting phusion protein.

Materials needed:

Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
The strain used is named EQ458. It is located in common species box; this is a Rosetta 2 (DE3) E. coli strain containing 6his-phusion-pET28 plasmid. The plasmid is KanR and the strain itself is CamR. The frozen stock is overnight growth of single colony. link to strain

LB medium. LB recipe
Chloramphenicol stock. Cam recipe
Kanamycin stock. Kan recipe
Refrigerated centrifuge.
Spectrophotometer and cuvettes.
FRENCH laboratory press. Georgiou lab, MBB, 3.310, ask before using.
IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
Disposable plastic columns. ThermoSci, cat #29922. Link to columns
Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml. Link to resin
Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730. Link to cassette

Lysis Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 40mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

Procedure: (for 2 x 500 mL cultures)

• Streak LB plate supplemented with Kan and Cam using frozen stock. Growth plate overnight at 37 C.
  
• Select single colony from O/N streak platen and inoculate 1.5 mL of LB broth supplemented with Kan and Cam.
  
• Grow overnight at 37 C shaking at 250 rpm.
  
• Use 500 uL of overnight culture to inoculate 500 mL of supplemented LB broth (in 2 L flask), grow as before for ~ 3-4 hours until an OD600 of between 0.4 and 0.6 is reached.
  
• Induce the cultures to express proteins by adding IPTG at a final concentration of 0.5 mM (2.5 mL per 500 mL) followed by overnight growth at 18 C, 250 rpm.
  
• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins.
  
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Collect and reintroduce into french press 1x.
  
• Heat denature at 70 C for about 15 mins.
  
• Spin down, 10,000 x g for 30 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  
• Syringe filter the SN.
  

Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel).

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 1x column volume of lysis buffer.
  
• Wash with 5x (column volume) of wash buffer.
  
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.
  

Dialysis: Link to dialysis cassette use

• Place dialysis cassette into storage buffer for 2 mins.
  
• Load dialysis cassette using a pipette or syringe.
  
• Squeeze the membrane to remove excess air.
  
• Replace top and place in beaker with 500 mL or 1 L of storage buffer. This should be done in the cold room on a stir plate.
  
• Allow to sit for 2 - 4 hours.
  
• Remove cassette and place in beaker with fresh storage buffer. Allow to sit overnight.
  
• If cassette has swollen, use syringe to remove some of the sample.
  
• Open top of cassette and remove sample.
  
• Store at -20 C.
  

Validation