Paul.xu at 23:32, 10 November 2011

2011-11-10T23:32:56Z

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| Refolding technique
| Advantages
| Disadvantages
|-
| Dialysis
| Simple
| Slow, Uses large volumes of buffer
|-
| Dilution
| Simple
| Slow, The protein is diluted to very low concentrations (10â50 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 Ã 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 Ã 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 ÃKTAprime 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 Ã 10 sec). 3. Centrifuge at 10000 Ã 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: ÃKTApurifier

[[Category:Protocols|Protocols]]

Protein Purification from Inclusion Bodies - Revision history

Paul.xu at 23:32, 10 November 2011