Refolding Record:
| Protein | |
|---|---|
| Protein Name | Isocitrate dehydrogenase |
| Abbreviated Name | IsoCIA |
| SCOP Family | Monomeric isocitrate dehydrogenase |
| Structure Notes | |
| Organism | Lingulodinium polyedrum |
| UniProt Accession | EC 1.3.99.1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 736 |
| Molecular Weight | 82331.1 |
| Pi | 5.83581 |
| Molecular Weight | 82331.1 |
| Disulphides | Unknown |
| Full Sequence |
mqitytltde spalatysflpivkaflsrahigvktsdislsgrilatfs eylkeeqrcedalellgelvkrsdanlikt pnisasipqlkaaikelqdkgymlpnypde pkndeelqiktkyqkvlgsavnpvlrqgnsdrrstkavkdyaknnpyrvv efnpnsktrvsymkegdffsnekavlidqdcvanieftsvdgkkeilkeglklekneild atfmdvqklqefyakeikaskdddvlfslhlkatmmkvsdpilfgyavkvffkelfiefq defeklginpnnglsellskiensskkdeilkkyseilaksadismvnsdkgitnlhvps dvivdasmpamlkngarlwdkegkekdtnavipdqtyatiyeaviedlhk ngtlnpsklgsvsnvglmakkaqeygshdktfvakeegtf kivsngkvll ehkvrkgdiyranqakfdavlnwidlgierselsgaeaifwldskrasnkimitlvqnrl kekgknvailtpkeaclrsleliregkdvisitgnvlrdyltdlfpilelgtsakmlsvv pmlnggamfetgaggsapkqveqlveenhlrwdslgeflalqaslefyan kcgnhkakvlaecldeaigewlennkapsrkvkeddnrtshfylamyfan
hlarqasdmelqsffkdialelssneekiraefndaqgvkvdlggyykfddekankimrp
721 satfnailek igqr
|
| 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 |