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 |
QSVPYGVSQI KAPALHSQGY TGSNVKVAVI DSGIDSSHPD LKVAGGASMV PSETNPFQDN NSHGTHVAGT VAALNNSIGV LGVAPSASLY AVKVLGADGS GQYSWIINGI EWAIANNMDV INMSLGGPSG SAALKAAVDK AVASGVVVVA AAGNEGTSGS SSTVGYPGKY PSVIAVGAVD SSNQRASFSS VGPELDVMAP GVSIQSTLPG NKYGAYNGTS MASPHVAGAA ALILSKHPNW TNTQVRSSLE NTTTKLGDSF YYGKGLINVQ AAAQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hayashi T, Matsubara M, Nohara D, Kojima S, Miura K, Sakai T (1994) FEBS Letters, 350, 109-112 |
| 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 | Column refolding: protein immobilization |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl pH 2.4 |
| Refolding Buffer | 2M potassium acetate, pH 6.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The protein was immobilized onto agarose beads, then denatured in solubilization buffer for 4h at 25degC. The protein was then refolded for 24h at 25degC in refolding buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 80% |
| Purity | n/a |
| Notes | Protein refolded at much higher yield (80%) when immobilized compared to freely diluted protein (28.3%). Lithium chloride was also used instead of potassium acetate with some success as well |