Refolding Record:
| Protein | |
|---|---|
| Protein Name | metalloprotease also know as Zinc metalloproteinase/disintegrin |
| Abbreviated Name | MT |
| SCOP Family | Blood coagulation inhibitor (disintegrin) |
| Structure Notes | |
| Organism | Agkistrodon halys brevicaudus (Korean slamosa snak |
| UniProt Accession | Q90WC0 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 292 |
| Molecular Weight | 32184.4 |
| Pi | 5.37 |
| Molecular Weight | 32184.4 |
| Disulphides | 3 |
| Full Sequence |
PAHQRYIELVIVADHGMFTKYNGDSDKIREWVRQMVNTVDEIYSYMYIDVALAGLEIWSNEDLINVQPAAPHTLDSFGKWRERDLLHRIHHDNAMLLTAIDFDGPTIGLAYVGTMCKPKGSTGVVQDHSTINLRVAVTMAHEIGHNLGIHHDTGSCSCGGYSCIMSPVISHEPSKYFSDCSYTQCWDFIMNQKPQCILNKPPLRTDTVSTPVSGNELLEAGEECDCGSPGNPCCDAATCKLRQGAQCAEGLCCDQCRFMKEGTICRRARGDDLDDYCNGISAGCPRNPFHA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jeon OH, Kim DS. (1999) Eur J Biochem., 263, 526-533 |
| Project Aim | Cloning and expression |
| Fusion | C-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 | 37.0 |
| Expression Time | 6 h |
| Expression Vector | pET22b |
| Expression Protocol | wo separate expression plasmids were constructed: pET-22b-MT-d-I, that contains cDNA encoding residues 190–486 and includes both metalloprotease and disintegrin domains; and pET-22b-MT-d-II that contains cDNA encoding residues 190–408 and has the metalloprotease domain only. The MT-d cDNAs were amplified by PCR, the amplified product purified by agarose gel electrophoresis and cloned into the pGEM™-T Easy Vector (Promega). To generate fusion protein specifying C-terminal His-tagged product, the insert from the pGEM™-T Easy Vector was isolated as a NdeI–HindIII fragment and cloned into pET22b (Novagen). An initiation codon was inserted at the N-terminus of the metalloprotease domain at an NdeI restriction site. A histidine hexamer was added to the C-terminus of the protein to facilitate purification of the recombinant protein using Ni–NTA resin (Quiogen). Each of the constructed plasmids was transformed into E. coli strain BL21 (DE3) pLysS, and grown in Luria–Bertani medium containing 200 mg·mL−1 ampicillin at 37 °C to a cell density of A600 = 0.8–1.0. The recombinant protein was induced by adding 1 m m isopropyl thio-β- d-galactopyranoside (IPTG) and incubation was continued for 6 h at 37 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8-1.0 = 600 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | Tris/EDTA buffer |
| Solubilization Buffer | 8 m urea containing 10 m m Tris/HCl (pH 8.0) and 100 m m NaCl/Pi. |
| Refolding Buffer | n/a |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were collected by centrifugation and suspended in lysis buffer (50 m m Tris/HCl pH 8.0, 100 m m NaCl, 1 m m EDTA, 0.1% Triton X-100). After disruption by freeze–thawing, cells were lysed by sonication. Cell lysate was centrifuged and washed three times with Tris/EDTA buffer. The induced protein was recovered as insoluble inclusion bodies. The precipitate was collected and solublized in 8 m urea containing 10 m m Tris/HCl (pH 8.0) and 100 m m NaCl/Pi. The solubilized protein was mixed with Ni–NTA resin (Qiagen) equilibrated with the same buffer. After incubation for 2 h at 4 °C, unbound protein was washed out with 6 m urea containing 10 m m Tris/HCl (pH 5.9) and 100 m m sodium phosphate. Bound protein was eluted with 6 m urea containing 10 m m Tris/HCl (pH 4.5) and 100 m m sodium phosphate, and the pH of the recovered protein solution was adjusted to 7.5. The purified protein (250 µg) was refolded by passing through a Sephadex G-25 desalting column (1 × 60 cm, Pharmacia) equilibrated with NaCl/Pi. Removal of the urea in this step was performed by eluting the protein at a flow rate of 0.1 mL·min−1. |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |