Refolding Record:
| Protein | |
|---|---|
| Protein Name | Neutrophil collagenase |
| Abbreviated Name | MMP8 |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P22894 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.100-262 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 163 |
| Molecular Weight | 18096.8 |
| Pi | 4.63824 |
| Molecular Weight | 18096.8 |
| Disulphides | 1 |
| Full Sequence |
MLTPGNPKWER TNLTYRIRNY TPQLSEAEVE RAIKDAFELW SVASPLIFTR ISQGEADINI AFYQRDHGDN SPFDGPNGIL AHAFQPGQGI GGDAHFDAEE TWTNTSANYN LFLVAAHEFG HSLGLAHSSD PGALMYPNYA FRETSNYSLP QDDIDGIQAI YG
|
| Notes | Selenomethionine-labeled protein |
| Expression | |
|---|---|
| Report | Qoronfleh MW, Ho TF, Brake PG, Banks TM, Pulvino TA, Wahl RC, Eshraghi J, Chowdhury SK, Ciccarelli RB, Jones BN (1995) J Biotechnology, 39, 119-128 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DL41 |
| Expression Temp | 37.0 |
| Expression Time | 1-3h |
| Expression Vector | pET11a |
| Expression Protocol | Cells were grown at 37degC in defined LeMaster medium devoid of methionine in the presence of 100microg/ml ampicillin. The medium was modified by adding Kao and Michayluk vitamin solution trace metal mix, and increasing glucose concentration to 0.2%. The enriched LeMaster medium contained L-selenomethionine at 25microg/ml. When A600 of the culture reached 0.7 expression was induced with 0.5mM IPTG. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M urea |
| Refolding Buffer | contains Zn2+ and Ca2+ |
| Pre-Refolding Purification | Ion-exchange chromatography |
| 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 | Inclusion bodies were recovered from lysed bacteria. The pellet was solubilized in 6M urea and centrifuged. The supernatant was then loaded onto a MonoQ sepharose column. Pooled fractions renatured by 10-fold dilution at 4degC. After the protein was concentrated, it was purified further using a Sepharcryl S-100 HR column. |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |