Refolding Record:
| Protein | |
|---|---|
| Protein Name | Matrix metalloproteinase 1 |
| Abbreviated Name | Dm1-MMP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Drosophila melanogaster (Fruit fly) |
| UniProt Accession | Q9GTK3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 567 |
| Molecular Weight | 63151.3 |
| Pi | 6.92 |
| Molecular Weight | 63151.3 |
| Disulphides | Unknown |
| Full Sequence |
MTNRRASGATHCKTTNNCNISNNSNKMTNCQSSVFIVVGTLFSILAAAQSAPVSTTTQAEIYLSQFGYLPASARNPASSGLHDQRTWVSAIEEFQSFAGLNITGELDAETMKLMSLPRCGVRDRVGTGDSRSKRYALQGSRWRVKNLTYKISKYPKRLKRVDVDAEIGRAFAVWSEDTDLTFTRKTSGPVHIEIKFVESEHGDGDAFDGQGGTLAHAFFPVFGGDAHFDDAELWTIGSPRGTNLFQVAAHEFGHSLGLSHSDQSSALMAPFYRGFEPVFKLDEDDKAAIQSLYGRKTNQLRPTNVYPATTQRPYSPPKVPLDDSICKDSKVDTLFNSAQGETYAFKGDKYYKLTTDSVEEGYPQLISKGWPGLPGNIDAAFTYKNGKTYFFKGTQYWRYQGRQMDGVYPKEISEGFTGIPDHLDAAMVWGGNGKIYFFKGSKFWRFDPAKRPPVKASYPKPISNWEGVPNNLDAALKYTNGYTYFFKGDKYYRFHDARFAVDSATPPFPRPTAHWWFGCKNTPSSTGNIVEGSDNEFEQHSMIPHADDGNGDDFDAGEWDRLSGSFV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Llano E, Pendás AM, Aza-Blanc P, Kornberg TB, López-Otín C (2000) Biologycal Chemistry, 17, 35978-35985 |
| Project Aim | Structural Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 30.0 |
| Expression Time | 5-20 h |
| Expression Vector | pRSET |
| Expression Protocol | Expression, Refolding, and Purification of Dm1-MMP-- A 735-bp fragment of the Dm1-MMP cDNA containing the pro- and catalytic domains was generated by PCR amplification with primers 5\'-CGGGATCCGCAATCGGCACCCGTTTCCACC (BamHI-proDm1) and 5\'-CGGAATTCATACAGTGACTGGATGGCCGC (EcoRI-proDm1) using the full-length Dm1-MMP cDNA as template. PCR amplification was performed for 30 cycles using the ExpandTM High Fidelity PCR system. Due to the design of the oligonucleotides, the amplified fragment could be cleaved at both ends with EcoRI and BamHI and ligated in frame into the pRSETB E. coli expression vector (Invitrogen) thereby adding an NH2-terminal His6 tag to the protein. The resulting pRSET-proDm1 vector was transformed into BL21(DE3)pLysS E. coli cells, and expression was induced by addition of isopropyl-1-thio--D-galactopyranoside (0.5 mM final concentration) followed by further incubation for 3-20 h at 30 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 mM Tris buffer, pH 7.6, containing 6 M GdnHCl, and 5 mM DTT |
| Refolding Buffer | 50 mM Tris buffer, pH 7.6, containing 5 mM CaCl2, 200 mM NaCl, 50 µM ZnSO4, 0.05% Brij 35, 20% glycerol, and 2 M GdnHCl, and then against the same buffer with 2 mM DTT, without GdnHCl. |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 2 mM |
| Refolding Protocol | Recombinant protein obtained in inclusion bodies was solubilized using 20 mM Tris buffer, pH 7.6, containing 6 M GdnHCl, and 5 mM DTT, and purified in a Superdex-75 column (Amersham Pharmacia Biotech) equilibrated with 20 mM Tris buffer, pH 7.6, containing 3 M GdnHCl, and 5 mM DTT. After SDS-PAGE analysis, peak fractions with the recombinant protein were pooled, and the GdnHCl concentration was adjusted to 6 M. Refolding was achieved by dialysis, first against a 50 mM Tris buffer, pH 7.6, containing 5 mM CaCl2, 200 mM NaCl, 50 µM ZnSO4, 0.05% Brij 35, 20% glycerol, and 2 M GdnHCl, and then against the same buffer with 2 mM DTT, without GdnHCl. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol,Brij 35 |
| Additives Concentration | 20%-0.05% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |