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. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding- Immobilized Metal affinity chromatography (IMAC) |
| Wash Buffer | 50 mM Tris/HCl, pH 8 containing 10 mM β -mercaptoethanol, 0.1% Berol 185 and 0.3 M NaCl |
| 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 J (50 mM Tris/HCl pH 8 containing 10 mM β -mercaptoethanol, 25 µM ZnCl2 , 0.1% CHAPS and 0.3 M NaCl)) |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | 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 hromatography buffers. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | CHAPS |
| Additives Concentration | 0.1% |
| Refolding Yield | 0.6 |
| Purity | n/a |
| Notes | NS3 specific activity of protein indicated in refolding yield |