Refolding Record:
| Protein | |
|---|---|
| Protein Name | Serine protease/NS3 protein/helicase NS3 |
| Abbreviated Name | NS3P |
| SCOP Family | Viral proteases [50596] |
| Structure Notes | |
| Organism | Hepatitis C virus genotype 1b |
| UniProt Accession | P26662 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 631 |
| Molecular Weight | 67141.7 |
| Pi | 6.98 |
| Molecular Weight | 67141.7 |
| Disulphides | Unknown |
| Full Sequence |
APITAYSQQTRGLLGCIITSLTGRDKNQVDGEVQVLSTATQSFLATCVNGVCWTVYHGAGSKTLAGPKGPITQMYTNVDQDLVGWPAPPGARSMTPCTCGSSDLYLVTRHADVVPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPSGHVVGIFRAAVCTRGVAKAVDFIPVESMETTMRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIEPNIRTGVRTITTGGPITYSTYCKFLADGGCSGGAYDIIICDECHSTDSTTILGIGTVLDQAETAGARLVVLATATPPGSITVPHPNIEEVALSNTGEIPFYGKAIPIEAIKGGRHLIFCHSKKKCDELAAKLTGLGLNAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRAQRRGRTGRGRSGIYRFVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVRLRAYLNTPGLPVCQDHLEFWESVFTGLTHIDAHFLSQTKQAGDNLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMACMSADLEVVT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Li M, Poliakov A, Danielson UH, Su Z, Janson JC. (2003) Biotechnol Lett, 25, 1729-1734 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | 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).In the other procedure urea was added to the French pressure cell homogenate before centrifugation to a concentration of 8 M followed by stirring for 4 h at 4 ◦ C. After centrifugation at 18 000 × g for 40 min, the supernatant was collected and imidazole was added to 50 mM. The sample, 60 ml, was applied at 4 ◦ C to a column filled with 10 ml Chelating Sepharose Fast Flow saturated with Ni2+ and equilibrated in 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.3 M NaCl, 4 M urea and 50 mM imidazole. Elution was performed by applying an imidazole concentration gradient up to 500 mM in the equilibration buffer. The sample obtained after this partial purification step was marked as sample 2 (S2). Protein concentration was measured according to Bradford using the protein assay kit from Bio-Rad with Coomassie Brilliant Blue G250 as dye reagent and bovine serum albumin as standard. 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. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | column refolding: ion-exchange chromatography |
| Wash Buffer | 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaClv |
| Solubilization Buffer | 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaCl, 8 M urea |
| Refolding Buffer | buffer D (50 mM Tris/HCl, pH 9 containing 10% (v/v) glycerol, 10 mM β -mercaptoethanol, 25 µM ZnCl2 and 0.1% CHAPS) |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.38 mg/ml |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | 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. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | CHAPS |
| Additives Concentration | 0.1% |
| Refolding Yield | 0.5 |
| Purity | n/a |
| Notes | NS3 specific activity of protein indicated in refolding yield |