Editing Protein Purification from Inclusion Bodies

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| Refolding technique 
| Advantages 
| Disadvantages
|-
| Dialysis 
| Simple 
| Slow, Uses large volumes of buffer
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| Dilution 
| Simple 
| Slow, The protein is diluted to very low concentrations (10â50 ÎŒg/ml)
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| Gel filtration<br> &nbsp; 
| 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<br> Refolding and purification in a single operation 
| Requires histidine-tagged protein
|-
| Ion exchange chromatography 
| Fast and simple<br> Straightforward to automate 
| Additives with opposite charge<br> 
to the ion exchanger should be<br> avoided<br> &nbsp; 

|}

'''Washing'''<br> Wash buffer: PBS containing 1% Triton X-100 and 1 mM EDTA, pH 7.4.<br> 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.<br> 2. Centrifuge at 10 000 to 30 000 Ã g for 10 to 30 min.<br> 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.<br> 4. Collect the pellet, which contains the inclusion bodies. 

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

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

'''Refolding Methods''' 

Examples:<br> '''I. Gel filtration'''<br> 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<br> glutathione (GSSG).<br> 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'''<br> Resuspension buffer: 20 mM Tris-HCl, pH 8.0<br> Isolation buffer: 2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton-X 100, pH 8.0<br> Binding buffer (port A1):&nbsp; 6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM&nbsp; imidazole, 1 mM 2-mercaptoethanol, pH 8.0<br> Solubilization buffer (port A2):&nbsp; 6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,&nbsp; 1 mM 2-mercaptoethanol, pH 8.0<br> Elution buffer (port A3):&nbsp; 20 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole,&nbsp; 1 mM 2-mercaptoethanol, pH 8.0<br> Refolding buffer (port B):&nbsp; 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,&nbsp; 1 mM 2-mercaptoethanol, pH 8. 

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

'''Solubilization and sample preparation'''<br> 1. Resuspend the inclusion body pellet in 5 ml of binding buffer.<br> 2. Stir for 30 to 60 min at room temperature.<br> 3. Centrifuge for 15 min at high speed, 4°C.<br> 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.<br> 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'''<br> 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.<br> 2. Place the three brown waste tubings in waste.<br> 3. Connect the column between port 1 on the injection valve (7-port valve) and the UV flow cell.<br> 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.<br> 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.<br> Note: If a Superloop is needed, additional information is supplied in the instructions for Superloop.<br> Application to use:'''On-Column Refolding HisTrap'''<br> 1 to 40 min: Binding (port A1)<br> 40 to 120 min: Refolding (port A1/B)<br> 120 to 140 min: Elution (Port A3/B)<br> Column binding capacity /ml medium<br> HisTrap FF, Approx 40 mg his-tagged protein<br> HisTrap HP,&nbsp; Approx 40 mg his-tagged protein 

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

2.'''Cation-Exchange Column''' 

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

[[Category:Protocols|Protocols]]