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 | High Pressure |
| Wash Buffer | n/a |
| Solubilization Buffer | n/a |
| Refolding Buffer | 50 mM Tris at pH 8.0, 4 mM GSSG, 2 mM DTT, 157 mM NaCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.5 mg/ml |
| Refolding Time | 24 |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 4/2 mM |
| Refolding Protocol | Pressure refolding Aggregated bikunin at a concentration of 0.5 mg/mL in various solutions was placed in heat-sealed 1-mL polypropylene syringes (enabling pressure transfer) along with 4 mM GSSG and 2 mM DTT to provide a redox environment for disulfide huffling. Samples were slowly pressurized (over 10 min) to minimize system heating and were then held at the desired temperature and pressure. Two different depressurization protocols were used. For slow depressurization, pressures were reduced by 5% of the initial pressure every 30 min until a pressure 20% of the initial pressure was reached. From this point, pressure was reduced by 10% (of the initial pressure) every 30 min until atmospheric pressure was obtained (about 8 h). Rapid depressurization occurred over a few seconds, when the pressurized system was opened to atmosphere. After depressurization, SEC-HPLC or RP-HPLC was conducted while the remaining samples were stored frozen at –20°C until further testing with SEC-HPLC, RP-HPLC, GEMMA, and activity assays. Freeze–thaw studies conducted at Bayer verified that monomer structure was unaffected by multiple freeze–thaw cycles (data not shown). In addition, no significant changes in refolding yields and chromatograms were seen before and after freezer storage through SEC-HPLC and RP-HPLC. Refolding yield was determined by dividing the monomer mass fraction by the total protein concentration (monomer and aggregate) and then converting the result to a percentage. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 65-75% |
| Purity | n/a |
| Notes | n/a |