Refolding Record:
| Protein | |
|---|---|
| Protein Name | Non-structural protein 1 |
| Abbreviated Name | NS1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Dengue virus type 2 |
| UniProt Accession | P29990 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 353 |
| Molecular Weight | 39939.5 |
| Pi | 6.4 |
| Molecular Weight | 39939.5 |
| Disulphides | Unknown |
| Full Sequence |
DSGCVVSWKNKELKCGSGIFITDNVHTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPELNHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPTELKYSWKTWGKAKMLSTESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLKEKQDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKNCHWPKSHTLWSNGVLESEMIIPKNLAGPVSQHNYRPGYHTQITGPWHLGKLEMDFDFCDGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRGEDGCWYGMEIRPLKEKEENLVNSLVTA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Huang JL, Huang JH, Shyu RH, Teng CW, Lin YL, Kuo MD, Yao CW, Shaio MF. (2001) J Med Virol., 65, 553-560 |
| Project Aim | Diagnostic studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2-3 h |
| Expression Vector | pET21b |
| Expression Protocol | The NS1 expression plasmids were transformed to BL21(DE3). Expression of the NS1 clone was induced by the addition of 1 mM isopropyl b-D-thiogalactopyranpside (IPTG) to a growing culture for 2 ± 3 hr. The bacteria were harvested and resuspended in TE buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA). After treatment with lysozyme and Triton X-100, the bacterial lysate was sonicated and centrifuged to pellet down the inclusion bodies, which were then further puri®ed by repeated centrifugation and washing. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 1 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl, 0.1 mM PMSF, 2.0 mM reduced glutathione, 0.2 mM oxidized glutathionethe |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,2/0.2 mM |
| Refolding Protocol | The insoluble inclusion bodies were denatured with 8 M urea. The solubilized proteins were then refolded by slowly dialyzing them out of the denaturant with refolding buffers (1 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl, 0.1 mM PMSF, 2.0 mM reduced glutathione, 0.2 mM oxidized glutathione) containing sequentially decreased concentrations of urea (4 M, 2 M, 1 M, 0 M). Finally, the inclusion bodies were completely solubilized in 1 PBS. The recombinant NS1 fragments were further puri®ed with T7-Tag af®nity puri®cation chromatography (Novagen, Madison, WI). The puri®ed and refolded proteins were analyzed with protein assay reagent (Bio- Rad, Hercules, CA) and SDS-PAGE to determine the protein concentration and purity. The purity was quanti®ed by CCD software (AlphaEase Version 2.02, Alpha Innotech, San Leandro, CA). |
| Refolding Assay | Western Blot,ELISA,postrefolding'analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | |