Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human Collagenase-3 (MMP-13) |
| Abbreviated Name | Collagenase-3 |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P45452 |
| 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 | 478 |
| Molecular Weight | 53819.8 |
| Pi | 5.32365 |
| Molecular Weight | 53819.8 |
| Disulphides | 0 |
| Full Sequence |
MHPGVLAAFLFLSWTHCRALPLPSGGDEDDLSEEDLQFAERYLRSYYHPTNLAGILKENA
ASSMTERLREMQSFFGLEVTGKLDDNTLDVMKKPRCGVPDVGEYNVFPRTLKWSKMNLTY
RIVNYTPDMTHSEVEKAFKKAFKVWSDVTPLNFTRLHDGIADIMISFGIKEHGDFYPFDG
PSGLLAHAFPPGPNYGGDAHFDDDETWTSSSKGYNLFLVAAHEFGHSLGLDHSKDPGALM
FPIYTYTGKSHFMLPDDDVQGIQSLYGPGDEDPNPKHPKTPDKCDPSLSLDAITSLRGET
MIFKDRFFWRLHPQQVDAELFLTKSFWPELPNRIDAAYEHPSHDLIFIFRGRKFWALNGY
DILEGYPKKISELGLPKEVKKISAAVHFEDTGKTLLFSGNQVWRYDDTNHIMDKDYPRLI
EEDFPGIGDKVDAVYEKNGYIYFFNGPIQFEYSIWSNRIVRVMPANSILWC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Pathak N, Hu SI, Koehn JA (1998) Protein Expression and Purification, 14, 283-288 |
| 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 | 30.0 |
| Expression Time | 16h |
| Expression Vector | pET3d |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 100mM Tris, 5 mM CaCl2, 10 mM DTT, 6 M urea, pH 7.5 |
| Refolding Buffer | 50mM Tris, 400mM NaCl, 10 mM CaCl2, 0.1 mM Zn(OAc)2, 5 mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 30 min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet, containing inclusion bodies from the overexpression of cd-CLN3, was resuspended in 25 ml of denaturation buffer (100 mM Tris, 5 mM CaCl2, 10 mM DTT, 6 M urea, pH 7.5) using a Dounce homogenizer and stirred for 1 h at room temperature. The pellet was collected at 100,000g for 1 h. The supernatant, containing urea-extracted proteins, was rapidly diluted at room temperature to a final volume of 400 ml using renaturation buffer (50 mM Tris, 10 mM CaCl2, 0.1 mM Zn(OAc)2, 5 mM DTT, pH 7.5) containing 400 mM NaCl and protease inhibitors. After the initial dilution, a small amount of precipitate formed and was collected at 35,000g for 30 min. Renaturation was completed by diafiltration in renaturation buffer using a LabScale TFF System (Millipore) equipped with a BioMax membrane (5000 MWCO). During this process, an additional 100-fold dilution was achieved. The soluble, renatured proteins were buffer exchanged in cation-exchange buffer (50mM MES, 5 mM CaCl2, 0.1 mM Zn(OAc)2, 5 mM DTT, pH 6.5) and the volume reduced using the LabScale TFF system in preparation of the chromatography step. A large amount of precipitation occurred during each of these stages and was collected at 35,000g for 30 min. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 25% |
| Purity | 40% |
| Notes | |