| 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 |
| Lysozyme |
| None |
| insoluble |
| Column refolding- Immobilized Metal affinity chromatography (IMAC) |
| 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaCl |
| 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaCl, 8 M urea |
| buffer J (50 mM Tris/HCl pH 8 containing 10 mM β -mercaptoethanol, 25 µM ZnCl2 , 0.1% CHAPS and 0.3 M NaCl)) |
| None |
| no tag |
| 8.0 |
| 25.0 |
| n/a |
| n/a |
| Beta-mercaptoethanol |
| 10 mM |
| For the IMAC refolding processes, a Chelating Sepharose Fast Flow column (5 ml) was equilibrated with 50 mM Tris/HCl pH 8 containing 10 mM β - mercaptoethanol, 25 µM ZnCl2 , 0.3 M NaCl, 4 M urea for S2, 8 M urea for S1 (buffer I). After sample loading, the protein was refolded by introducing a gradient from buffer I to buffer J (50 mM Tris/HCl pH 8 containing 10 mM β -mercaptoethanol, 25 µM ZnCl2 , 0.1% CHAPS and 0.3 M NaCl) in four column volumes. The refolded NS3 was finally eluted by introducing a gradient from buffer J to buffer K (buffer J plus 0.5 M imidazole) in three column volumes. S2 should be desalted before being loaded to the IEC and IMAC columns. For this purpose, a 5 ml Sephadex G-25 column was equilibrated and eluted with the initial chromatography buffers.
|
| Activity assay |
| None |
| CHAPS |
| 0.1% |
| 0.9 |
| n/a |
| NS3 specific activity of protein indicated in refolding yield |