| 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 |
| Gel filtration |
| 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 |
| 50 mM Tris/HCl pH 8 containing 1 M urea, 10 mM β -mercaptoethanol, 25 µM ZnCl2 , 0.2 M NaCl and 0.1% CHAPS (buffer A) |
| None |
| no tag |
| 8.0 |
| 25.0 |
| 0.38 mg/ml |
| n/a |
| Beta-mercaptoethanol |
| 10 mM,10 mM |
| All buffers used in this study are specified in Table 1. In gel filtration experiments without gradient, the columns were equilibrated and eluted with 50 mM Tris/HCl pH 8 containing 1 M urea, 10 mM β -mercaptoethanol, 25 µM ZnCl2 , 0.2 M NaCl and 0.1% CHAPS (buffer A). In gel filtration experiments using gradients, before sample loading, the column was first equilibrated in buffer A followed by a 4 ml or 12 ml gradient from buffer A to buffer B. For the elution of sample S2, the composition of buffer B was 50 mM Tris/HCl, pH 8 plus 10 mM β - mercaptoethanol, 25 µM ZnCl2 , 0.2 M NaCl and 4 M urea. For sample S1 the urea concentration of buffer B was increased to 8 M. Thus S1 was both loaded and eluted in the presence of buffer B containing 8 M urea. |
| Activity assay |
| None |
| None,CHAPS |
| 0.1% |
| 0.6 |
| n/a |
| NS3 specific activity of protein indicated in refolding yield |