Refolding Record:
| Protein | |
|---|---|
| Protein Name | Non-structural protein 1 |
| Abbreviated Name | NS1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | West Nile virus (WNV |
| UniProt Accession | P06935 |
| 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 | 39608.7 |
| Pi | 5.42 |
| Molecular Weight | 39608.7 |
| Disulphides | Unknown |
| Full Sequence |
DTGCAIDIGRQELRCGSGVFIHNDVEAWMDRYKFYPETPQGLAKIIQKAHAEGVCGLRSVSRLEHQMWEAIKDELNTLLKENGVDLSVVVEKQNGMYKAAPKRLAATTEKLEMGWKAWGKSIIFAPELANNTFVIDGPETEECPTANRAWNSMEVEDFGFGLTSTRMFLRIRETNTTECDSKIIGTAVKNNMAVHSDLSYWIESGLNDTWKLERAVLGEVKSCTWPETHTLWGDGVLESDLIIPITLAGPRSNHNRRPGYKTQNQGPWDEGRVEIDFDYCPGTTVTISDSCEHRGPAARTTTESGKLITDWCCRSCTLPPLRFQTENGCWYGMEIRPTRHDEKTLVQSRVNA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chung KM, Nybakken GE, Thompson BS, Engle MJ, Marri A, Fremont DH, Diamond MS. (2006) J Virol, 80, 1340-1351 |
| Project Aim | Vaccine studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | For expression of WNV NS1 fragments in E. coli, plasmids were transformed into BL21 Codon Plus E. coli cells (Stratagene). Bacteria were grown in LB, induced with 0.5 to 1.0 mM isopropyl thiogalactoside, pelleted, lysed after the addition of lysozyme, and sonicated, and NS1 fragments were recovered as insoluble aggregate from the inclusion bodies. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine hydrochloride and 20 mM β-mercaptoethanol |
| Refolding Buffer | 0.4 M l-arginine, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione |
| Pre-Refolding Purification | not specified |
| 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 | 5/0.5 mM |
| Refolding Protocol | Fragments were solubilized in the presence of 6 M guanidine hydrochloride and 20 mM β-mercaptoethanol and refolded by rapidly diluting out the solubilizing reagents in the presence of 0.4 M l-arginine, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione. Refolded NS1 fragments were separated from aggregates on Superdex 75 or 200 16/60 size exclusion columns (Amersham Biosciences) and concentrated using a Centricon 10 spin column in 20 mM HEPES (pH 7.4). |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.4 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |