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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 268 |
| Molecular Weight | 30150.7 |
| Pi | 6.75802 |
| Molecular Weight | 30150.7 |
| Disulphides | 1 |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSA D YLSQYGYLQK PLEGSNNFKP EDITEALRAF QEASELPVSG QLDDATRARM RQPRCGLEDP FNQKTLKYLL LGRWRKKHLT FRILNLPSTL PPHTARAALR QAFQDWSNVA PLTFQEVQAG
AADIRLSFHG RQSSYCSNTF DGPGRVLAHA DIPELGSVHF DEDEFWTEGT YRGVNLRIIA AHEVGHALGL GHSRYSQALM APVYEGYRPH FKLHPDDVAG IQALYGKKS
|
| Notes | Two sequence mutants also produced: E88P/P90V: MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSA D YLSQYGYLQK PLEGSNNFKP 50 EDITEALRAF QEASELPVSG QLDDATRARM RQPRCGLPDV FNQKTLKYLL 100 LGRWRKKHLT FRILNLPSTL PPHTARAALR QAFQDWSNVA PLTFQEVQAG 150 AADIRLSFHG RQSSYCSNTF DGPGRVLAHA DIPELGSVHF DEDEFWTEGT 200 YRGVNLRIIA AHEVGHALGL GHSRYSQALM APVYEGYRPH FKLHPDDVAG 250 IQALYGKKS Number of amino acids: 268 Molecular weight: 30120.6 Theoretical pI: 6.98 C166S MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSA D YLSQYGYLQK PLEGSNNFKP 50 EDITEALRAF QEASELPVSG QLDDATRARM RQPRCGLEDP FNQKTLKYLL 100 LGRWRKKHLT FRILNLPSTL PPHTARAALR QAFQDWSNVA PLTFQEVQAG 150 AADIRLSFHG RQSSYCCNTF DGPGRVLAHA DIPELGSVHF DEDEFWTEGT 200 YRGVNLRIIA AHEVGHALGL GHSRYSQALM APVYEGYRPH FKLHPDDVAG 250 IQALYGKKS Number of amino acids: 268 Molecular weight: 30166.7 Theoretical pI: 6.76 |
| 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 | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a,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 | |
| Purity | |
| Notes | |