| Dilution/Column Refolding Combination |
| Phosphate buffered saline (PBS), 25% sucrose, 5mM EDTA, 1% Triton X-100 |
| 50mM TrisCl pH 8.0, 5M GdnHCl, 5mM EDTA |
| 50mM TrisCl pH 8.0, 20% glycerol, 1mM DTT, 1microg/ml pepstatin, 20microg/ml aprotinin |
| None |
| no tag |
| 8.0 |
| 4.0 |
|
| 12-16h |
| DTT |
| 1mM |
| 1g of E.coli cells were resupsended in 50ml of 20mM TrisCl pH 7.5, 20% sucrose, 1mM EDTA. after 10min incubation on ice, cells were centrifuged (4000xg) and the pelleted cells were resuspended in 50ml ice-cold water for 10min. The cells were centrifuged again (8000xg) and the pellet resuspended in 10ml buffer P (PBS with 5mM EDTA, 1 microg/ml pepstatin, 20microg/ml aprotinin). The suspension was sonicated (3x30sec) in an ice water bath, RNaseT1 (1300u) and DNaseI (500microg) were added and the mixture was incubated at room temperature for 10min. The suspension was diluted by the addition of 40ml Buffer P and the inclusion bodies were collected by centrifugation (13000g x 30min). The inclusion bodies were then resupsended in 40ml Wash buffer, incubated on ice for 10min and centrifuged (10min x 25000g). This wash step was repeated three times and then inclusion bodies were finally resuspended in 10ml solubilization buffer. The suspension was sonicated briefly for 5sec, then incubated on ice for an additional hour. Following centrifugation, (30g x 12000) the supernatant was added to 100ml refolding buffer and stirred gently at 4degC overnight. The soluble (S) and insoluble (I) fractions were separated by centrifugation (30min x 13500g) and the soluble fraction was concentrated to 1ml and dialyzed against Buffer A (50mM Na phosphate, 1mM DTT, 20% glycerol, 1mM EDTA, 1microg/ml pepstatin, 1mM benzamidine) for 3h at 4degC. The dialyzed protein was then centrifuged (14000rpm) and the supernatant loaded onto an S-sepharose fast flow column and eluted with a step NaCl gradient (50-1000mM) in buffer A. The protein eluted at 300mM NaCl.
The insoluble refolded fraction (I) was dissolved in 10ml of 6M GdnHCl in Buffer B (50mM TrisCl pH 8, 1mM DTT, 2mM EDTA). The solution was then centrifuged (10000g x 5min) and the supernatant was passed through two Superose 12 columns attached in tandem equilibrated in buffer B containing 4M GdnHCl. Fractions containing the protein were pooled and dialyzed against buffer C (4M urea, 50mM sodium phosophate pH 6.5, 1mM benzamidine, 1microg/ml pepstatin, 4mM EDTA) in a recirculating tangential flow ultrfiltration system. The dialyzed fraction was filtered (0.22micron) and then passed through a MonoS column. After washing (Buffer C), the protein was eluted with a gradient of 0-500mM NaCl in Buffer C. |
| Ultraviolet (UV) Absorbance |
| None |
| None,Glycerol |
| 20% |
| 0.5mg/g packed cells |
|
| Only 10% of protein in original inclusion bodies refolded in initial dilution refolding (fraction S), most protein successfully produced from Fraction I (0.5mg/ml packed cells). |