Refolding Record:
| Protein | |
|---|---|
| Protein Name | Nucleocapsid protein |
| Abbreviated Name | Protein N |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human SARS coronavirus |
| UniProt Accession | P59595 |
| 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 | 422 |
| Molecular Weight | 46025.0 |
| Pi | 10.1 |
| Molecular Weight | 46025.0 |
| Disulphides | Unknown |
| Full Sequence |
MSDNGPQSNQRSAPRITFGGPTDSTDNNQNGGRNGARPKQRRPQGLPNNTASWFTALTQHGKEELRFPRGQGVPINTNSGPDDQIGYYRRATRRVRGGDGKMKELSPRWYFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLNQLESKVSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDNVILLNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAADMDDFSRQLQNSMSGASADSTQA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Mark J, Li X, Cyr T, Fournier S, Jaentschke B, Hefford MA. (2008) Biochemical and Biophysical Research Com, 1, 1 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 1.0 |
| Expression Time | 1 |
| Expression Vector | pQE |
| Expression Protocol | SARS N-protein expression and purification. Escherichia coli M15 and BL21(DE3) cells, transformed with pQE-2NP (pQE-2 expressing the N-protein), were used to produce protein as previously described [17] and [19]. Purification was performed under denaturing conditions using a His-trap HP metal affinity column (GE Lifesciences). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | n/a |
| Refolding Buffer | 10 mM Tris, 100 mM sodium phosphate, 150 mM NaCl, and 8 M urea, pH 8.0 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 1.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The purified N-protein was transferred to dialysis tubing (7500 MWCO) and dialyzed into a urea-supplemented refolding buffer (10 mM Tris, 100 mM sodium phosphate, 150 mM NaCl, and 8 M urea, pH 8.0). Urea was then gradually removed by a stepwise replacement of the buffer with Tris/phosphate buffer (10 mM Tris, 100 mM sodium phosphate, and 150 mM NaCl, pH 8.0) containing decreasing concentrations of urea (8, 4, 2, 1, 0.5, and 0 M). In samples where the N-terminal (His)6-tag was removed, the pH of the buffer was adjusted to 7.0 by dialysis and Qiagen DAPase (dipeptidase) added to the protein solution. |
| Refolding Assay | SDS-PAGE,Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Refolding Temperature not stated |