Refolding Record:
| Protein | |
|---|---|
| Protein Name | Succinate dehydrogenase |
| Abbreviated Name | SucA |
| SCOP Family | Succinate dehydrogenase |
| Structure Notes | |
| Organism | Lingulodinium polyedrum |
| UniProt Accession | EC 1.3.99.1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 631 |
| Molecular Weight | 69291.0 |
| Pi | 5.88793 |
| Molecular Weight | 69291.0 |
| Disulphides | Unknown |
| Full Sequence |
mwrcvsrgfr apasktsslf dgvsgsrfsr ffstgstdtr ssytivdhty davvvgaggaglraaiglse hgfntacitk lfptrshtva aqgginaalg nmseddwrwh mydtvkgsdwlgdqdaiqym creapkavie lenyglpfsr teegkiyqra fggqsldfgk ggqayrcacaadrtghallh tlygqamkhn tqffveyfal dllmasdgsc qgvialnmedgtlhrfrssqtilatggygr ayfsatsaht ctgdgnamva raglplqdle fvqfhptgiy gagclitegsrgeggilrns egerfmerya ptakdlasrd vvsrsmtmei regrgvgphkylhlnhlppevlkerlp gisetaaifa gvdvtkepip vlptvhynmg giptnyhgev vtikgddpdavipglmaage aacasvhgan rlganslldivvfgracanr vaeiskpgek qkplekdagektiawldrlr nsngslptst irlnmqrimq nnaavfrtqe tleegcqlid kawesfgdvqvkdrsmiwns dlietlelen llinasitmh saearkesrg aharedftkr edgewmkhtlgywedekvrl dyrpvhmdtl ddeidtfppk arvy
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hidetoshi Akimoto, Tomoya Kinumi and Yoshihiro Ohmiya (2005) J Biological Rhythms, 20, 479-489 |
| Project Aim | Evolutionary Studies |
| Fusion | N-terminal hexahistidine tag |
| 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 | 3hrs |
| Expression Vector | pCR T7/NT |
| Expression Protocol | The E. coli BL21(DE3)cells transformed with pHis--sucA, were cultured to mid-log phase and induced by 1mM IPTG at 37C for 3h. The induced cells were harvested; resuspended in 100mM Tris-HCl buffer, pH 8.0; and disrupted using nitrogen pressure apparatus. After centrifugation the pellet was briefly washed twice with 20mM phosphate buffer, pH7.4. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Nitrogen Pressure |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Phosphate buffer pH 7.4 |
| Solubilization Buffer | 20mM phosphate buffer, 8M urea, 1mM mercaptoethanol, 0.5M NaCl 10mM imidazole |
| Refolding Buffer | 20mM Tris-HCl pH 8.0 1mM mercaptoethanol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | The isolated inclusion bodies were solubilized in 20 mM phosphate buffer containing 8 M urea, 1mM?-mercaptoethanol, 0.5MNaCl, and10 mM imidazole and were centrifuged. The supernatant was filtered through a 0.22-?m filter (Millipore, Bedford, MA)and then passed through a HiTrap Chelating HPcolumn (AmershamBiosciences, Piscataway, NJ). Bound proteins were purified following the manufacturer’s protocols. Refolding of the denatured protein was achieved by serial dialysis at 4° C in 20mM Tris-HCl buffer,pH8.0, containing 1 mM ?-mercaptoethanol, then in the same buffer with- out ?-mercaptoethanol. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |