Refolding Record:
| Protein | |
|---|---|
| Protein Name | Matrix metalloprotease 13 |
| Abbreviated Name | MMP13 |
| 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 | n |
| Domain | aa.1-248 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 289 |
| Molecular Weight | 32750.7 |
| Pi | 5.65316 |
| Molecular Weight | 32750.7 |
| Disulphides | 1 |
| Full Sequence |
MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWIRPRDLQ
MHPGVLAAFL FLSWTHCRAL PLPSGGDEDD LSEEDLQFAE RYLRSYYHPT NLAGILKENA ASSMTERLRE MQSFFGLEVT GKLDDNTLDV MKKPRCGVPD VGEYNVFPRT LKWSKMNLTY RIVNYTPDMT HSEVEKAFKK AFKVWSDVTP LNFTRLHDGI ADIMISFGIK EHGDFYPFDG PSGLLAHAFP PGPNYGGDAH
FDDDETWTSS SKGYNLFLVA AHEFGHSLGL DHSKDPGALM FPIYTYTG
|
| Notes | 2 different refolding protocols described in this paper Only aa.1-248 of MMP-13 expressed, refolded and purified in this paper. But construct also had a 41-residue N-terminal extension containing His6-tag. |
| Expression | |
|---|---|
| Report | Hardern IM, Knauper V, Ernill RJ, Taylor IWF, Cooper KL, Abbott WM (2000) Protein Expression and Purification, 19, 246-252 |
| Project Aim | Folding |
| 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)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 26h |
| Expression Vector | pRSETC |
| Expression Protocol | 20L of HYE20 medium containing 3.0g/L KH2PO4, 6g/L Na2HPO4, 0.5g/L NaCl, 2.0g/L casein hydrolysate, 10.0g/L (NH4)2SO4, 35g/L glycerol, 20g/L yeast extract, 0.5g/L MgSO4.7H2O, 0.0294g/L CaCl2.2H2O, 0.008g/L thiamine, 40mg/L FeSO4, 20mg/mL citric acid, 100microg/ml ampicillin, 34microg/ml chloramphenicol and trace elements, pH 6.7. The culture was inoculated with 600ml seed culture grown overnight in L broth at 37degC to OD550=3. The 20L culture was grown to OD550=19, and protein expression was induced by the addition of 1mM IPTG. Cells were harvested at various timepoints up to 26h after induction by centrifugation (30min, 3500g) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 550 = 19 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris, 100mM NaCl, 0.005% Brij 35, pH 8.0 |
| Solubilization Buffer | 6M urea, 20mM Tris, 1mM 2-mercaptoethanol pH 7.5 |
| Refolding Buffer | 20mM Tris-H2SO4, 20% glycerol, 100mM Na2SO4, 0.5microM ZnCl2, 0.005%(w/v) Brij 35 pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 18h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 200g of cell paste was thawed and resuspended in 2L of wash buffer containing 1microg/ml leupeptin and pepstatin, 1mM PMSF and 1mM EDTA using a homogenizer. The cells were lysed by two passes through a cell disrupter and were centrifuged (30min x 10000g). The pellet was resuspended in 500ml of wash buffer and centrifuged again. This washing step was repeated a further two times. Finally the pellet was resuspended in 500ml wash buffer and frozen at -80degC in 10ml aliquots. 10ml of inclusion body preparation was thawed and centrifuged (10000g, 30min) and then resuspended to a total volume of 25ml in solubilization buffer using a homogenizer. Solubilization was allowed to occur at room temperature for 60min with mixing. The mixture was then centrifuged (10000g) and the supernatant retained. The solubilized protein was divided into two portions and each half was refolded by dilution into a vortexing 7L volume of refolding buffer 1 at a flow rate of 1ml/min. The dilute protein solution was allowed to stand at 4degC for 18h before each 7L volume was loaded onto a separate 20ml Ni-NTA agarose column equilibrated in refold buffer. After washing of the column in refold buffer and nickel wash buffer (20mM Tris, 5mM CaCl2, 150mM NaCl, 0.005%(w/v) Brij 35, pH 8.0), the protein was eluted in nickel wash buffer containing 100mM imidazole. The protein was then dialyzed against 3 x 1h x 5L changes of nickel wash buffer before being centrifuged (30min, 10000g). The clarified supernatants were then pooled. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 90% |
| Purity | |
| Notes | |