Refolding Record:
| Protein | |
|---|---|
| Protein Name | Citrate synthase |
| Abbreviated Name | CS |
| SCOP Family | Citrate synthase |
| Structure Notes | |
| Organism | Pig (Sus scrofa) |
| UniProt Accession | P00889 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 438 |
| Molecular Weight | 48919.2 |
| Pi | 7.0 |
| Molecular Weight | 48919.2 |
| Disulphides | 0 |
| Full Sequence |
ASSTNLKDILADLIPKEQARIKTFRQQHGNTVVGQITVDMMYGGMRGMKGLVYETSVLDPDEGIRFRGYSIPECQKMLPKAKGGEEPLPEGLFWLLVTGQIPTEEQVSWLSKEWAKRAALPSHVVTMLDNFPTNLHPMSQLSAAITALNSESNFARAYAEGIHRTKYWELIYEDCMDLIAKLPCVAAKIYRNLYREGSSIGAIDSKLDWSHNFTNMLGYTDAQFTELMRLYLTIHSDHEGGNVSAHTSHLVGSALSDPYLSFAAAMNGLAGPLHGLANQEVLVWLTQLQKEVGKDVSDEKLRDYIWNTLNSGRVVPGYGHAVLRKTDPRYTCQREFALKHLPHDPMFKLVAQLYKIVPNVLLEQGKAKNPWPNVDAHSGVLLQYYGMTEMNYY
TVLFGVSRALGVLAQLIWSRALGFPLERPKSMSTDGLIKLVDSK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Daugherty DL, Rozema D, Hanson PE, Gellman SH (1998) Biologycal Chemistry, 18, 33961-33971 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M GdmCl and 40 mM DTT |
| Refolding Buffer | 0.024 mg/ml CS, 0.4 mM DTT, 60 mM GdmCl, 100 mM Tris-Cl, 0.5 mM EDTA, 0.4 mM detergent, and 4.8 mM B-cyclodextrin |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.4 mM,0.4 mM |
| Refolding Protocol | n a typical GdmCl denaturation/refolding study, 2.4 mg/ml CS (as a suspension in 3.2 M ammonium sulfate) was dissolved in 6 M GdmCl and 40 mM DTT and allowed to stand for 1 h. The CS solution was then diluted by a factor of 70 with Tris-chloride buffer containing detergent and EDTA to give concentrations of 0.034 mg/ml CS, 0.57 mM DTT, 86 mM GdmCl, 143 mM Tris-Cl (pH 7.6), 0.71 mM EDTA, and 0.57 mM detergent. After 1 h, 30 ul of 16 mM -cyclodextrin stock solution was added to 70 ul of the above solution to bring the final concentrations to 0.024 mg/ml CS, 0.4 mM DTT, 60 mM GdmCl, 100 mM Tris-Cl, 0.5 mM EDTA, 0.4 mM detergent, and 4.8 mM B-cyclodextrin. This solution also contained 100 mM ammonium sulfate from the original suspension; when CS was desalted before use, refolding yields were not substantially changed (desalted CS could not be stored for long periods). After sitting overnight, the solutions were assayed for enzymatic activity. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | β-cyclodextrin |
| Additives Concentration | 4.8 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |