Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tissue inhibitor of metalloproteinase |
| Abbreviated Name | TIMP-1 |
| SCOP Family | Tissue inhibitor of metalloproteinases, TIMP |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P12032 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 208 |
| Molecular Weight | 22628.3 |
| Pi | 9.13757 |
| Molecular Weight | 22628.3 |
| Disulphides | 6 |
| Full Sequence |
MMAPFASLASGILLLLSLIASSKACSCAPPHPQTAFCNSDLVIRAKFMGSPEINETTLYQ
RYKIKMTKMLKGFKAVGNAADIRYAYTPVMESLCGYAHKSQNRSEEFLITGRLRNGNLHI
SACSFLVPWRTLSPAQQRAFSKTYSAGCGVCTVFPCLSIPCKLESDTHCLWTDQVLVGSE
DYQSRHFACLPRNPGLCTWRSLGAR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cocuzzi ET, Walther SE, Rajan S, Denhardt DT. (1992) FEBS Letters, 307, 375-378 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 30.0 |
| Expression Time | 4h |
| Expression Vector | pET3c |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 8M urea, 50mM Tris-HCl, 0.1M betamercaptoethanol, pH 7.5 |
| Refolding Buffer | 50mM Tris-HCl |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.01mg/ml |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The target protein was purified from solubilized inclusion bodies on Bio-Rex 70 ion exchange equilibbrated with 8M urea, 50mM Tris-HCl, pH 7.5. Betamercaptoethanol was supplemented into the fractions containing pure target protein to a final concentration of 140mM. At the stage the buffer consisted 50mM Tris-HCl, 140mM betamercaptoethanol, 6M urea, pH 7.5. The protein was diluted to 0.2mg/ml in the same buffer and cystamine was added to 292mM. This solution was rocked at 4C overnight. The urea was slowly diluted to 0.3M with 50mM Tris-HCl, pH 9.0 and the solution was exposed to the air at 4C for 24h without stirring. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 0.5% |
| Purity | |
| Notes | |