Refolding Record:
| Protein | |
|---|---|
| Protein Name | Stefin B |
| Abbreviated Name | Stefin B |
| SCOP Family | Cystatins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P04080 |
| 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 | 99 |
| Molecular Weight | 11139.6 |
| Pi | 6.96224 |
| Molecular Weight | 11139.6 |
| Disulphides | 0 |
| Full Sequence |
MMCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVG
DEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Thiele U, Auerswald EA, Gebhard W, Assfalg-Machleidt I, Popovic T, Machleidt W. (1988) Biol Chem Hoppe Seyler., 369, 1167-1178 |
| Project Aim | Structure-Function |
| Fusion | N-terminal MS-2 polymerase |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | 537 |
| Expression Temp | 42.0 |
| Expression Time | 7h |
| Expression Vector | pEx31A |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 100mM Tris-HCl, 10mM EDTA, 5mM benzamidine, 5mM betamercaptoethanol, 2M urea, pH 7.5 |
| Solubilization Buffer | 50mM Tris-HCl, 10mM betamercaptoethanol, 6M urea, pH 8.5 |
| Refolding Buffer | 50mM Tris-HCl, 10mM betamercaptoethanol |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were washed twice in 100mM Tris-HCl, 10mM EDTA, 5mM benzamidine, 5mM betamercaptoethanol, 2M urea, pH 7.5, prior to solubilization. The solubilized protein was purified on a Sephacryl S-300 column in 50mM Tris-HCl, 6M guanidinium chloride, 20mM betamercaptoethanol pH 8.5. The recovered pure protein was precipitated by dialysis with water and recovered by centrifugation. The fusion protein was treated with CNBr (1000-fold molar excess) in 75% formic acid. Subsequently the protein was dialysed against 50mM Tris-HCl, 10mM betamercaptoethanol, 6M urea, pH 8.5 and refolded by dialysis against the same buffer without urea. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 15% |
| Purity | pure |
| Notes | |