Refolding Record:
| Protein | |
|---|---|
| Protein Name | Torsin-1A Also known as: Dystonia 1 protein |
| Abbreviated Name | DYT1, TOR1A |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O14656 |
| 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 | 313 |
| Molecular Weight | 35777.2 |
| Pi | 6.16 |
| Molecular Weight | 35777.2 |
| Disulphides | Unknown |
| Full Sequence |
VEPISLGLALAGVLTGYIYPRLYCLFAECCGQKRSLSREALQKDLDDNLFGQHLAKKIILNAVFGFINNPKPKKPLTLSLHGWTGTGKNFVSKIIAENIYEGGLNSDYVHLFVATLHFPHASNITLYKDQLQLWIRGNVSACARSIFIFDEMDKMHAGLIDAIKPFLDYYDLVDGVSYQKAMFIFLSNAGAERITDVALDFWRSGKQREDIKLKDIEHALSVSVFNNKNSGFWHSSLIDRNLIDYFVPFLPLEYKHLKMCIRVEMQSRGYEIDEDIVSRVAEEMTFFPKEERVFSDKGCKTVFTKLDYYYDD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Konakova M, Pulst SM. (2004) J Mol Neurosci., 25, 105-117 |
| 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 | Origami B(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pET44a |
| Expression Protocol | Protein Expression, Refolding, and Purification OrigamiB (DE3) cells (Novagen), carrying the appropriate constructs, were grown in Luria- Bertani broth containing 50 µg/mLcarbenicillin, 15 µg/mLkanamycin, and 12.5 µg/mLtetracycline at 37ºC to an A600of 0.6–0.88 and induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 5h. Cells were collected by centrifugation, washed with 20 mMTris-HCl (pH 8.0), and resuspended in 0.1 culture volume of lysis buffer (20 mMTris-HCl at pH 7.5, 10 mMEDTA, 1% Triton X-100), supplemented with 100 µg/mLlysozyme. After a 15-min incubation at 30ºC, bacterial cells were disrupted by sonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.88 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mMTris-HCl (pH 8.0) |
| Solubilization Buffer | 0.3% N-lauroylsarcosine, 50 mM3-(cyclohexylamino)-propanesulfonic acid, and 1 mMdithiothreitol (DTT) (pH 11) |
| Refolding Buffer | 20 mMTris-HCl (pH 9.0), 1 mM reduced glutathione, and 0.2 mM oxidized glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 36 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.2 mM |
| Refolding Protocol | Inclusion bodies were harvested by centrifugation at 10,000gfor 10 min and washed several times with lysis buffer. Finally, the pellets were washed with 20 mMTris-HCl (pH 8.0) and stored at –20ºC. Inclusion bodies proteins were solubilized in 0.3% N-lauroylsarcosine, 50 mM3- (cyclohexylamino)-propanesulfonic acid, and 1 mMdithiothreitol (DTT) (pH 11) at room temper- ature with gentle stirring overnight. Prior to refolding, the protein concentration of the samples was adjusted to 0.5 mg/mLwith solubilization buffer. Detergent was removed by dialysis against 20 mM Tris-HCl, 0.1 mMDTT (pH 8.5)m, for 18 h, followed by dialysis against 20 mMTris-HCl (pH 8.5) for 36 h at 4ºC. Oxidation of the recombinant proteins was achieved by dialysis against 20 mMTris-HCl (pH 9.0), 1 mMreduced glutathione, and 0.2 mM oxidized glutathione for 18 h at 4ºC. Finally, the proteins samples were dialyzed against 20 mMTris-HCl (pH 7.5) and 300 mMNaCl for 24 h at 4ºC and cleared by centrifugation at 20,000gfor 30 min. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |