Refolding Record:
| Protein | |
|---|---|
| Protein Name | cytochrome c |
| Abbreviated Name | Cyt c |
| SCOP Family | monodomain cytochrome C |
| Structure Notes | |
| Organism | Horse (Equus caballus) |
| UniProt Accession | P00004 |
| 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 | 105 |
| Molecular Weight | 11701.5 |
| Pi | 9.58 |
| Molecular Weight | 11701.5 |
| Disulphides | Unknown |
| Full Sequence |
GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITW
KEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Shimojo K, Oshima T, Naganawa H, Goto M (2007) Biomacromolecules, 8, 3061-3066 |
| 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 | Liquid-Liquid Extraction |
| Wash Buffer | n/a |
| Solubilization Buffer | 10 mM Tris buffer, 10 mM MES buffer, and HCl solution containing 8 M urea |
| Refolding Buffer | Oct6CH2COOH 1 mM in isooctane + 10 volume % 1- octanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 30.0 |
| Protein Concentration | n/a |
| Refolding Time | 12 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Liquid-Liquid Extraction of Denatured Cyt c Using Calixarene. Native Cyt c (10 M) was dissolved in denaturant solutions, the pH of which was adjusted from 2 to 7 using 10 mM Tris buffer, 10 mM MES buffer, and HCl solution containing 8 M urea,60-62 and incubated overnight at room temperature. An extracting phase was prepared by dissolving tOct[6]CH2COOH (1 mM) in isooctane containing 10 vol % 1-octanol, which assists the solubilization of tOct[6]CH2COOH.63 Equal volumes of the aqueous and organic solutions were mixed and gently shaken at 30 C for 12 h to attain equilibrium. After phase separation, the concentrations of Cyt c in both phases were determined by the absorbance of the Soret band peak using a UV-vis spectrophotometer (JASCO U-best 570), which then allowed calculation of the extractability (E = [Cyt c]org,eq/[Cyt c]aq,ini; org, aq, eq, and ini denote the organic phase, the aqueous phase, the equilibrium state, and the initial state, respectively). The concentration of urea extracted in the organic phase was directly measured by the urease-indophenol assay using a urea nitrogen assay kit. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Calixarrene |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |