Refolding Record:
| Protein | |
|---|---|
| Protein Name | Adrenodoxin |
| Abbreviated Name | Adx |
| SCOP Family | 2Fe-2S ferredoxin-related |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P00257 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | proadrenodoxin |
| Chain Length | 203 |
| Molecular Weight | 21422.2 |
| Pi | 5.31 |
| Molecular Weight | 21422.2 |
| Disulphides | 0 |
| Full Sequence |
MAARLLRVASAALGDTAGRWRLLARPRAGAGGLRGSRGPGLGGGAVATRTLSVSGRAQSSSEDKITVHFINRDGETLTTKGKIGDSLLDVVVQNNLDIDGFGACEGTLACSTCHLIFEQHIFEKLEAITDEENDMLDLAYGLTDRSRLGCQICLTKAMDNMTVRVPDAVSDARESIDMGMNSSKIPAANKAREAELAAATAEE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bera, A. K., Bernhardt, R. (1999) Archives Biochemistry and Biophysics, 367, 89-94 |
| Project Aim | Folding,Functional Studies |
| Fusion | C-terminal sequence |
| 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 | 3h |
| Expression Vector | pET-3s |
| Expression Protocol | Cells were grown in NB medium containing ampicillin. After induction cells were lysed and lysate centrifuged (15,000 rpm/min, 4degC. 15min) to obtain inclusion bodies. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.9 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM TrisHCl, pH 7.5, 100mM NaCl, 2mM DTT |
| Solubilization Buffer | 6M GuHCl, 50mM TrisHCl, pH 7.5, 2mM DTT |
| Refolding Buffer | 50mM TrisHCl, 10mM MgCl2, 25 mM KCl, 1 mM DTT |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Inclusion bodies were solubilized at 250microM in solubilization buffer. This then underwent a 100 fold dilution with refolding buffer in the presence of 4microM GroEL(final concentration after dilution). The refolded protein solution was then centrifuged(15,000g, 15min, 4degC) before being loaded onto a Sephacryl S 400 column. |
| Refolding Assay | Ligand Binding,Folding Kinetics |
| Refolding Chaperones | GroEL minichaperone |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |