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 | 552 |
| Molecular Weight | 62397.4 |
| Pi | 6.55679 |
| Molecular Weight | 62397.4 |
| Disulphides | 1 |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRTGSDLDICSWY
MNCQQLWLGF LLPMTVSGRV LGLAEVAPVD YLSQYGYLQK PLEGSNNFKP EDITEALRAF QEASELPVSG QLDDATRARM RQPRCGLEDP FNQKTLKYLL LGRWRKKHLT FRILNLPSTL PPHTARAALR QAFQDWSNVA PLTFQEVQAG AADIRLSFHG RQSSYCSNTF DGPGRVLAHA DIPELGSVHF DEDEFWTEGT
YRGVNLRIIA AHEVGHALGL GHSRYSQALM APVYEGYRPH FKLHPDDVAG IQALYGKKSP VIRDEEEEET ELPTVPPVPT EPSPMPDPCS SELDAMMLGP RGKTYAFKGD YVWTVSDSGP GPLFRVSALW EGLPGNLDAA VYSPRTQWIH FFKGDKVWRY INFKMSPGFP KKLNRVEPNL DAALYWPLNQ KVFLFKGSGY
WQWDELARTD FSSYPKPIKG LFTGVPNQPS AAMSWQDGRV YFFKGKVYWR LNQQLRVEKG YPRNISHNWM HCRPRTIDTT PSGGNTTPSG TGITLDTTLS ATETTFEY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Pendas AM, Knauper V, Puente XS, Llano E, Mattei M-G, Apte S, Murphy G, Lopez-Otin C (1997) J Biol Chem, 272, 4281-4286 |
| Project Aim | Structure-Function |
| 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 | pRSETA |
| Expression Protocol | Cells were grown in 2TY medium at 37degC, when A600 reached 0.6, expression was induced by the addition of 0.5mM IPTG and cells were incubated a further 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 | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris pH 8.0, 6M urea, 5mM DTT |
| Refolding Buffer | 20mM Tris/HCl pH 8.0, 5mM CaCl2, 50microM ZnCl2 |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | 5mM,5mM |
| Refolding Protocol | Inclusion bodies were solubilized in solubilization buffer, then purified by gel filtration chromatography using a Sephacryl S-200 column pre-equilibrated in 50mM Tris/HCl pH 8.0, 4M urea, 5mM DTT. Selected fractions were then combined and refolded by dilution (1:10) into refolding buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |