Refolding Record:
| Protein | |
|---|---|
| Protein Name | Matrix metalloprotease 19 |
| Abbreviated Name | MMP19 |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | MMP_HUMAN |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | C166S |
| Chain Length | 268 |
| Molecular Weight | 30166.7 |
| Pi | 6.75 |
| Molecular Weight | 30166.7 |
| Disulphides | 1 |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSA D YLSQYGYLQK PLEGSNNFKP EDITEALRAF QEASELPVSG QLDDATRARM RQPRCGLEDP FNQKTLKYLL LGRWRKKHLT FRILNLPSTL PPHTARAALR QAFQDWSNVA PLTFQEVQAG AADIRLSFHG RQSSYCCNTF DGPGRVLAHA DIPELGSVHF DEDEFWTEGT YRGVNLRIIA AHEVGHALGL GHSRYSQALM APVYEGYRPH FKLHPDDVAG IQALYGKKS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Stracke JO, Hutton M, Stewart M, Pendas AM, Smith B, Lopez-Otin C, Murphy G, Knauper V (2000) J Biol Chem, 275, 14809-14816 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahis + pro seq |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 30.0 |
| Expression Time | 3-20h |
| Expression Vector | pRSET B |
| Expression Protocol | Cells were grown in 2YT medium, when A600 reached 0.6, expression was induced with 0.5mM IPTG followed by further incubation for 3-20h at 30degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris/H2SO4 pH 8.0, 6M urea, 5mM 2-mercaptoethanol, 0.01% NaN3 |
| Refolding Buffer | 20mM Tris/H2SO4 pH 7.6, 100mM Na2SO4, 5mM CaSO4, 5microM ZnSO4, 5% glycerol, 0.03% Brij 35 (w/v), 0.01% NaN3 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were solubilized in solubilization buffer for 30min at 37degC. Refolding was performed by dilution (1:250) into refolding buffer at 4degC. The refolding mixture was filtered and applied to a Ni-NTA agarose column and the protein was purified. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |