Refolding Record:
| Protein | |
|---|---|
| Protein Name | Macrophage Metalloelastase (MMP-12) |
| Abbreviated Name | MMP-12 |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P39900 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Catalytic domain aa.106-268 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 163 |
| Molecular Weight | 18077.2 |
| Pi | 6.11373 |
| Molecular Weight | 18077.2 |
| Disulphides | 0 |
| Full Sequence |
GPVWR KHYITYRINN YTPDMNREDV DYAIRKAFQV WSNVTPLKFS KINTGMADIL VVFARGAHGD FHAFDGKGGI LAHAFGPGSG IGGDAHFDED EFWTTHSGGT NLFLTAVHEI GHSLGLGHSS DPKAVMFPTY KYVDINTFRL SADDIRGIQS LYGDPKEN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Parkar AA, Stow MD, Smith K, Panicker AK, Guilloteau JP, Jupp R, Crowe SJ (2000) Protein Expression and Purification, 20, 152-161 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET21a |
| Expression Protocol | Cells were cultured in baffled shake flasks at 37degC in Luria broth medium supplemented with 100 mg/ml ampicillin. Protein expression was induced when the cells had grown to an OD600 value of 0.6–0.8 by the addition of 0.5mM IPTG. Cells were harvested 4 h postinduction by centrifugation at 2000g for 20 min. The pelleted cells were resuspended in 20 ml of 100 mM Tris, pH 8.0, per liter of culture and stored frozen for up to several months before preparation of inclusion bodies. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Column Refolding Combination |
| Wash Buffer | 0.1% Triton X-100 |
| Solubilization Buffer | 50mM Tris-HCl, 30mM DTT, 8M guanidinium chloride pH8.0 |
| Refolding Buffer | 50mM HEPES, 10mM CaCl2, 0.1mM zinc acetate, 3 M urea |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.2 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.1-0.2 mg/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Fractions containing MMP-12 from the gel filtration chromatography column were pooled and diluted into refolding buffer (3 M urea, 50 mM Hepes, 10 mM CaCl2, 30 mM NaCl, 0.1 mM zinc acetate, pH 7.2) to achieve a final protein concentration of 0.1-0.2mg/ml during the refolding (typically 500 ml from a 1-liter preparation). The resultant diluted protein was dialyzed in a two-step procedure: a first dialysis against refolding buffer and a second dialysis against cation exchange buffer 1 (3 M urea, 10 mM Hepes, 2 mM CaCl2, pH 7.0). Each dialysis step was performed at 4degC for 24 h against eight volumes of the dialysis buffer and three buffer changes in each step. Cation exchange chromatography was conducted using SP-Sepharose resin (Amersham Pharmacia Biotech) in a batch binding procedure. A 6-ml bed volume of SP-Sepharose resin (prewashed with cation exchange buffer 1) was mixed with 500 ml of dialyzed protein from above and incubated at room temperature with rotation for 60 min. The protein-bound SP-Sepharose resin was collected by centrifugation and then progressively washed with the following buffer series: three washes with cation exchange buffer 1, one wash with cation exchange buffer 2 (2Murea, 10 mM Hepes, 2 mM CaCl2, pH 7.0), one wash with cation exchange buffer 3 (1Murea, 10 mM Hepes, 2 mMCaCl2, pH 7.0), two washes with cation exchange buffer 4 (10 mM Hepes, 2 mM CaCl2, pH 7.0). For each wash, the resin was incubated at room temperature with rotation for 10 min with 10 bed volumes of the relevant buffer. Bound MMP-12_CAT was eluted from the resin with five elutions, each of 8 ml, of elution buffer (10 mM Hepes, 2 mM CaCl2, 250 mM NaCl, pH 7.0). The elutions were pooled and stored frozen at -20degC until further use. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |