Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bikunin Placental |
| Abbreviated Name | HAI-2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O43291 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 225 |
| Molecular Weight | 25415.7 |
| Pi | 8.26 |
| Molecular Weight | 25415.7 |
| Disulphides | 6 |
| Full Sequence |
ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNNYLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMFNYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEACMLRCFRQQENPPLPLGSKVVVLAGLFVMVLILFLGASMVYLIRVARRNQERALRTVWSSGDDKEQLVKNTYVL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Seefeldt MB, Ouyang J, Froland WA, Carpenter JF, Randolph TW. (2004) Protein Science, 13, 2639-2650 |
| 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 | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M guanidine HCl and 12 mM DTT |
| Refolding Buffer | 50 mM Tris (pH 8.0), 157 mM NaCl, 4 mM GSSG, and 2 mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 4/2 mM,4/2 mM |
| Refolding Protocol | Aggregated bikunin was incubated at 37°C in the presence of 6M guanidine HCl and 12 mM DTT to reduce disulfide bonds and disassociate the aggregate (Clark et al. 1998). Two refolding protocols were employed, \\\"dialysis refolding\\\" and dilution refolding. Dialysis refolding was conducted by placing the denatured protein (0.5 mg/mL) in a dialysis cassette and submerging it in excess refolding buffer of 50 mM Tris (pH 8.0), 157 mM NaCl, 4 mM GSSG, and 2 mM DTT. The samples were dialyzed overnight. |
| Refolding Assay | RP-HPLC,Activity assay,SEC-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | \\\"Dialysis refolding\\\" resulted in increased aggregation (tetramer and larger by GEMMA analysis; data not shown) |