| Li M, Poliakov A, Danielson UH, Su Z, Janson JC.
(2003)
Biotechnol Lett,
25,
1729-1734 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| None |
| 0.0 |
| 00 |
| n/a |
| Expression and purification of the NS3 protein The details of the protocol for expression of the NS3 protein has previously been published (Poliakov et al. 2002). In this comparative study, two different sample preparation procedures were applied. In both of them an E. coli sediment from a 3 l culture was re-suspended in 40 ml 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol and 0.3 M NaCl and homogenized by two passages through a French pressure cell. In one of procedures the French pressure cell homogenate was directly centrifuged at 18 000 × g for 40 min. The sediment was washed three times with a washing buffer composed of 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 (a UV-transparent, non-ionic detergent from Berol Nobel, Stenungsund, Sweden, possessing a HLB-value similar to that of Triton X-100) and 0.3 M NaCl. The washed protein aggregates were then dissolved in denaturing buffer, i.e. washing buffer supplemented with 8 M urea. After removal of residual solids by centrifugation, the dissolved sample was marked as sample 1 (S1). |
| Not Stated |
| OD n/a =
n/a |
| French Press |
| Detergents |
| None |
| insoluble |
| column refolding: ion-exchange chromatography |
| 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaClv |
| 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaCl, 8 M urea |
| buffer D (50 mM Tris/HCl, pH 9 containing 10% (v/v) glycerol, 10 mM β -mercaptoethanol, 25 µM ZnCl2 and 0.1% CHAPS) |
| None |
| no tag |
| 9.0 |
| 25.0 |
| 0.82 mg/ml |
| n/a |
| Beta-mercaptoethanol |
| 10 mM |
| DEAE Sepharose Fast Flow was used in the IEC refolding experiments. The column (7 ml) was equilibrated with 50 mM Tris/HCl pH 9 containing 10 mM β -mercaptoethanol, 25 µM ZnCl2 , 4 M urea for S2, 8 M urea for S1 (buffer C). The sample from S1 was 0.5 ml at 0.82 mg protein ml−1 and the sample from S2 was 1 ml at 0.38 mg protein ml−1 . The flow-rate was 0.4 ml min−1 in all experiments. After the sample was loaded, refolding was initiated by gradually decreasing the urea concentration to 0 M. This was achieved by introducing a gradient from buffer C to buffer D (50 mM Tris/HCl, pH 9 containing 10% (v/v) glycerol, 10 mM β -mercaptoethanol, 25 µM ZnCl2 and 0.1% CHAPS) in five column volumes. Finally, the sample was eluted by introducing a gradient from buffer D to buffer E (buffer D plus 0.5 M NaCl) in two column volumes. This procedure was named the three-buffer system. In the two-buffer system procedure, the column was first equilibrated with the urea containing buffer C. After sample loading, the adsorbed protein was simultaneously refolded and eluted by introducing a gradient from buffer C to buffer E (buffer D + 0.5 M NaCl) in four column volumes. For comparison, the strong cation exchanger SP Sepharose Fast Flow (7 ml column) was used for IEC refolding at a lower pH. Here buffers C and D were replaced by buffers F and G containing 50 mM sodium acetate at pH 4 as the buffering salt. The other components were the same as used in the experiments with the DEAE Sepharose Fast Flow ion exchanger. The protein was refolded by introducing a gradient from buffer F to buffer G in five column volumes and subsequently eluted by introducing a gradient from buffer G to buffer H (buffer G plus 0.5 M NaCl) in two column volumes.
|
| Activity assay |
| None |
| CHAPS |
| 0.1% |
| 1 |
| n/a |
| NS3 specific activity of protein indicated in refolding yield |