Refolding Record:
| Protein | |
|---|---|
| Protein Name | Subtilisin BPN |
| Abbreviated Name | Subtilisin BPN |
| SCOP Family | Subtilases |
| Structure Notes | |
| Organism | Bacillus amyloliquefaciens |
| UniProt Accession | P00782 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 274 |
| Molecular Weight | 27462.6 |
| Pi | 6.3 |
| Molecular Weight | 27462.6 |
| Disulphides | 0 |
| Full Sequence |
QSVPYGVSQIKA PALHSQGYTG SNVKVAVIDS GIDSSHPDLK
VAGGASMVPS ETNPFQDNNS HGTHVAGTVA ALNNSIGVLG VAPSASLYAV KVLGADGSGQ YSWIINGIEW AIANNMDVIN MSLGGPSGSA ALKAAVDKAV ASGVVVVAAA GNEGTSGSSS TVGYPGKYPS VIAVGAVDSS NQRASFSSVG PELDVMAPGV SIQSTLPGNK YGAYNGTSMA SPHVAGAAAL ILSKHPNWTN TQVRSSLENT TTKLGDSFYY GKGLINVQAA AQ
|
| Notes | Mature protein without the pro-sequence refolded |
| Expression | |
|---|---|
| Report | Matsubara M, Kurimoto E, Kojima S, Miura K, Sakai T (1994) FEBS Letters, 342, 193-196 |
| 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 | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl pH 2.0 |
| Refolding Buffer | 2M potassium acetate, pH 6.5, 24-fold molar excess of mutant subtilisin inhibitor (SSI) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 1microg/ml |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Protein was unfolded in solubilization buffer for 2h at room temperature. Refolding was initiated by mixing the protein 100-fold with refolding buffer containing various salts at different concentrations, the final pH was 6.5. Protein was folded at either 4degC or 15degC for 24h |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 100% |
| Purity | n/a |
| Notes | Refolding analysed with a range of different organic salts, potassium acetate found to be the most effective. Without SSI, refolding yield was 30%, with SSI yield was 100%. Refolding experiments performed at 15degC and 4degC. |