Refolding Record:
| Protein | |
|---|---|
| Protein Name | Macrophage Metalloelastase (MMP-12) |
| Abbreviated Name | MMP-12 |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | Q63341 |
| 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 | 465 |
| Molecular Weight | 53738.4 |
| Pi | 8.73489 |
| Molecular Weight | 53738.4 |
| Disulphides | 1 |
| Full Sequence |
MKFLLVLVLL VSLQVSACGA APMNESEFAE WYLSRFFDYQ GDRIPMTKTK TNRNLLEEKL QEMQQFFGLE VTGQLDTSTL KIMHTSRCGV PDVQHLRAVP QRSRWMKRYL TYRIYNYTPD MKRADVDYIF QKAFQVWSDV TPLRFRKIHK GEADITILFA FGDHGDFYDF DGKGGTLAHA FYPGPGIQGD AHFDEAETWT KSFQGTNLFL VAVHELGHSL GLRHSNNPKS IMYPTYRYLH PNTFRLSADD IHSIQSLYGA PVKNPSLTNP GSPPSTVCHQ SLSFDAVTTV GDKIFFFKDW FFWWRLPGSP ATNITSISSM WPTIPSGIQA AYEIGGRNQL FLFKDEKYWL INNLVPEPHY PRSIHSLGFP ASVKKIDAAV FDPLRQKVYF FVDKQYWRYD
VRQELMDAAY PKLISTHFPG IRPKIDAVLY FKRHYYIFQG AYQLEYDPLL DRVTKTLSST SWFGC
|
| Notes | In this study, 2 different contructs of MMP-12 expressed and refolded - full intact protein and catalytic domain Catalytic domain: LRAVP 100 QRSRWMKRYL TYRIYNYTPD MKRADVDYIF QKAFQVWSDV TPLRFRKIHK 150 GEADITILFA FGDHGDFYDF DGKGGTLAHA FYPGPGIQGD AHFDEAETWT 200 KSFQGTNLFL VAVHELGHSL GLRHSNNPKS IMYPTYRYLH PNTFRLSADD 250 IHSIQSLYGA PVKNPSLTNP GSPPSTV Number of amino acids: 182 Molecular weight: 20785 Theoretical pI: 9.05 |
| Expression | |
|---|---|
| Report | Fu J-Y, Lyga A, Shi H, Blue M-L, Dixon B, Chen D (2001) Protein Expression and Purification, 21, 268-274 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL23(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 |
| Expression Vector | pET3a/pET14b |
| Expression Protocol | An overnight culture of cells in Luria broth was diluted 25x by the same media and incubated until OD600 reached 0.6-0.8. Expression was induced with 0.4mM IPTG and incubation continued for a further 3h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 50mM Hepes pH 7.5, 0.05% Brij35, 1mM PMSF, 100microg/ml DNase, 2000U/ml lysozyme |
| Solubilization Buffer | 7M GdnHcl |
| Refolding Buffer | 50mM Hepes pH 7.5, 0.05% Brij35, 150mM NaCl, 5mM CaCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet from 0.5L culture was suspended in 50ml wash buffer, homogenized and sonicated. After centrifugation (300g, 15min), the supernatant was further centifuged at 100000g for 30min. The pellet was resuspended in 50ml wash buffer and centrifuged again (100000g) before being resuspended in 10ml solubilization buffer and homogenized with a glass homogenizer. The sample was gently shaken for 60min at room temp and centrifuged (3000g, 20min). The supernatant was collected and loaded onto a Sephadex G-25 column for desalting and refolding, with elution in refolding buffer. The refolded protein was then purified further with a chelating sepharose-fast flow resin using ZnCl2 |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 3.6mg/L |
| Purity | |
| Notes | |