Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human Placental Bikunin |
| Abbreviated Name | Bikunin |
| SCOP Family | Small Kunitz-type inhibitors & BPTI-like toxins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O43291 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 229 |
| Molecular Weight | 25415.7 |
| Pi | 8.26353 |
| Molecular Weight | 25415.7 |
| Disulphides | 6 |
| Full Sequence |
ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTD
GSCQLFVYGGCDGNSNNYLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSE
DHSSDMFNYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEACM
LRCFRQQENPPLPLGSKVVVLAGLFVMVLILFLGASMVYLIRVARRNQERALRTVWSSGD
DKEQLVKNTYVL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Matthew B. Seefeldt, Jun Ouyang, Wayne A Froland, John F Carpenter, Theodore W Randolph (2004) Protein Science, 13, 2639-2650 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | CHO |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | unknown |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | High Pressure |
| Wash Buffer | none |
| Solubilization Buffer | none |
| Refolding Buffer | 50 mM Tris, 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 hrs |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Soluble aggregates of bikunin from CHO cell fermentation (Bayer Corp.) were diluted in refolding buffer and placed in a heat sealed 1 ml syringe. The sample was placed in a specialized high pressure chamber (BaroFold, Inc.) pressurized to 2000 bar and held for 24 hours. Pressure was reduced by 100 bar every 30 min until 400 bar then reduced 200 bar every 30 min. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 70 |
| Purity | |
| Notes | RP-HPLC assay was the same as activity assay. If initial protein concentration was reduced to 0.06 mg/ml %96 yields were obtained. Dilution refolding at 0.4 mg/ml resulted in a 55% yield. |