Protein Purification from Inclusion Bodies

Refolding technique	Advantages	Disadvantages
Dialysis	Simple	Slow, Uses large volumes of buffer
Dilution	Simple	Slow, The protein is diluted to very low concentrations (10–50 ÎŒg/ml)
Gel filtration	Straightforward to automate, Refolding and aggregate removal in a single operation	Sample volume is limiting
Immobilized metal ion affinity	Fast and simple chromatography (IMAC) Straightforward to automate Refolding and purification in a single operation	Requires histidine-tagged protein
Ion exchange chromatography	Fast and simple Straightforward to automate	Additives with opposite charge to the ion exchanger should be avoided

Washing
Wash buffer: PBS containing 1% Triton X-100 and 1 mM EDTA, pH 7.4.
1. Dilute the cell lysate in wash buffer, or add Triton X-100 and EDTA to concentrations of 1% and 1 mM, respectively. Lysis can also be done directly in the wash buffer.
2. Centrifuge at 10 000 to 30 000 × g for 10 to 30 min.
3. Save the pellet, suspend and wash twice with the same buffer and centrifuge at 10 000 to 30 000 × g for 10 to 30 min.
4. Collect the pellet, which contains the inclusion bodies.

Solubilization
50 mM Tris-HCl, pH 8.0, 8 M urea, at room temperature for 60 min

Refolding Additives
Urea 2M
Guanidinium hydrochloride 1M
Arginine 0.5 M
Glycerol 0.4 M
Sucrose 0.4 M
Lauryl maltoside 0.3 mM
Polyethylene glycol 3550 0.05% (w/v)
Tris 0.5 M
Triton X-100 10 mM
n-hexanol 5 mM
Salts 0.5 mM

Refolding Methods

Examples:
I. Gel filtration
Superdex 75 10/300 GL, use 8 M urea, 0.2 M DTT to dissolve protein, use refolding buffer: 0.1 M Tris-HCl, pH 8.7, 1 mM EDTA, 150 mM NaCl, 3 mM reduced glutathione (GSH)/0.3 mM oxidized
glutathione (GSSG).
Refolding: 6 ml linear gradient from 8 M to 2 M urea in refolding buffer

II. HisTrap (FF) column or HisTrap HP (1 ml) column on ÄKTAprime plus
Resuspension buffer: 20 mM Tris-HCl, pH 8.0
Isolation buffer: 2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton-X 100, pH 8.0
Binding buffer (port A1):  6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM  imidazole, 1 mM 2-mercaptoethanol, pH 8.0
Solubilization buffer (port A2):  6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,  1 mM 2-mercaptoethanol, pH 8.0
Elution buffer (port A3):  20 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole,  1 mM 2-mercaptoethanol, pH 8.0
Refolding buffer (port B):  20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,  1 mM 2-mercaptoethanol, pH 8.

Protocol
Disruption, wash, and isolation of inclusion bodies
1. Resuspend the cell paste from 100 ml culture in 4 ml resuspension buffer.
2. Disrupt cells with sonication on ice (e.g., 4 × 10 sec).
3. Centrifuge at 10000 × g for 10 min at 4°C.
4. Remove supernatant and resuspend pellet in 3 ml of cold isolation buffer. Sonicate as above.
5. Centrifuge at high speed for 10 min at 4°C.
6. Repeat steps 4 and 5.
At this stage the pellet material can be washed once in buffer lacking urea and stored frozen for later processing.

Solubilization and sample preparation
1. Resuspend the inclusion body pellet in 5 ml of binding buffer.
2. Stir for 30 to 60 min at room temperature.
3. Centrifuge for 15 min at high speed, 4°C.
4. Remove any remaining particles by passing the sample through a 0.45 ÎŒm filter.

The optimal concentration of 2-mercaptoethanol (0 to 20 mM) must be determined experimentally for each individual protein.
If it has not been prepared as above, adjust the sample to the composition of binding buffer by diluting in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26/10 Desalting, then pass the sample through a 0.45 ÎŒm filter.

FPLC set up
1. Place each inlet tubing from port A (8-port valve) in eluents as given above and the tubing from port B (2-port valve) in the elution buffer.
2. Place the three brown waste tubings in waste.
3. Connect the column between port 1 on the injection valve (7-port valve) and the UV flow cell.
4. Fill the fraction collector rack with 18-mm tubes (minimum 40) and position the white plate on the fractionation arm against the first tube.
5. Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve. Use a syringe to manually fill the loop.
Note: If a Superloop is needed, additional information is supplied in the instructions for Superloop.
Application to use:On-Column Refolding HisTrap
1 to 40 min: Binding (port A1)
40 to 120 min: Refolding (port A1/B)
120 to 140 min: Elution (Port A3/B)
Column binding capacity /ml medium
HisTrap FF, Approx 40 mg his-tagged protein
HisTrap HP,  Approx 40 mg his-tagged protein

III. Refolding using ion exchange chromatography
Example:
1. Anion-Exchange Column
Column: HiTrap Q FF, 1 ml
Protein: denatured-reduced (contains 17 disulfide bonds)
Start buffer: 50 mM Tris-HCl, 3 mM EDTA, 8 M urea, pH 8.5
Refolding Buffer: 50 mM Tris-HCl, 1 mM EDTA, 79 mM urea, 1.1 mM oxidized glutathione (GSSG), 2.2 mM reduced glutathione (GSH), pH 8.5
Incubation period: 0 to 40 h
Wash buffer: 50 mM Tris-HCl, 1 mM EDTA, pH 8.5
Elution buffer: 0 to 1M NaCl in 50 mM Tris HCl, pH 8.5

2.Cation-Exchange Column

Column: SP Sepharose HP, 5 ml
Sample: Lysozyme (8 mg)
Start Buffer: 50 mM Tris HCl, 6 M urea, 3 mM reduced glutathione (GSH)/0.3 mM oxidized glutathione (GSSG), pH 6.2
Elution buffer: 50 mM Tris HCl, 1 M urea, 300 mM NaCl, 3 mM GSH/0.3 mM GSSG, pH 10
Flow rate: 0.4 ml/min
Instrument: ÄKTApurifier