Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cyclophilin, Variant A |
| Abbreviated Name | Cyclophilin A |
| SCOP Family | Cyclophilin (peptidylprolyl isomerase) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P62937 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 164 |
| Molecular Weight | 17881.3 |
| Pi | 7.82 |
| Molecular Weight | 17881.3 |
| Disulphides | 0 |
| Full Sequence |
VNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Altamirano, M. M., Golbik, R., Zahn, R., Buckle, A. M., Fersht, A. R. (1997) Proc. Natl. Acad. Sci. USA, 94, 3576-3578 |
| Project Aim | Folding |
| 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 | n/a |
| Expression Vector | n/a |
| Expression Protocol | n/a |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Chaperone-assisted refolding |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M Urea, 0.1 M potassium phosphate, pH 7.8, 5 mM 2-mercaptoethanol |
| Refolding Buffer | 0.1 M potassium phosphate, pH 7.8, 5 mM 2-mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 30min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Suspension of 200 microlitres of gel (wet sedimented volume, either bound via the Ni-NTA linkage or covalently linked to minichaperone via CNBr activation) was mixed with refoding buffer to give a volume of 990 microlitres. Cyclophilin A (10 microlitres per 100 microM stock solution in solubilization buffer) was added to the suspension and mixed in an up and down mixer. Gel suspension then underwent centrifugation to obtain supernatant. The pellet was then washed in empty miniprep columns with the eluate then added to the supernatant of the previous centrifugation to obtain the refolded Cyclophilin A. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | GroEL minichaperone |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |