Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rhizopuspepsinogen |
| Abbreviated Name | Rhizopuspepsinogen |
| SCOP Family | Pepsin-like proteases |
| Structure Notes | |
| Organism | Rhizopus chinensis |
| UniProt Accession | P06026 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 373 |
| Molecular Weight | 39165.8 |
| Pi | 5.98 |
| Molecular Weight | 39165.8 |
| Disulphides | 2 |
| Full Sequence |
PGEKKISI PLAKNPNYKP SAKNAIQKAI AKYNKHKINT STGGIVPDAG VGTVPMTDYG NDVEYYGQVT IGTPGKKFNL
DFDTGSSDLW IASTLCTNCG SRQTKYDPKQ SSTYQADGRT WSISYGDGSS ASGILAKDNV NLGGLLIKGQ TIELAKREAA SFANGPNDGL LGLGFDTITT VRGVKTPMDN LISQGLISRP IFGVYLGKAS NGGGGEYIFG GYDSTKFKGS LTTVPIDNSR GWWGITVDRA TVGTSTVASS FDGILDTGTT LLILPNNVAA
SVARAYGASD NGDGTYTISC DTSRFKPLVF SINGASFQVS PDSLVFEEYQ GQCIAGFGYG NFDFAIIGDT FLKNNYVVFN QGVPEVQIAP VAQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chen Z, Koelsch G, Han HP, Wang XJ, Lin XL, Hartsuck JA, Tang J (1991) J Biol Chem, 266, 11718-11725 |
| 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 | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET3a |
| Expression Protocol | Cells were grown in LB medium at 37degC until A600 reached 0.5. 0.5mM IPTG was added and incubation continued for 3h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100mM Tris-HCl, 150mM NaCl, 1% Triton X-100 pH7.4 |
| Solubilization Buffer | 8M urea, 50mM CAPS, 1mM glycine, 1mM EDTA, 50 mM betamercaptoethanol, pH 10.5 |
| Refolding Buffer | H2O |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 9.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 2h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Harvested cells were resuspended in 100mM TrisHCl pH 7.4, 0.15M NaCl with 100mg lysozyme. The suspension was stirred for 30min at room temperature and frozen at -20degC overnight. After thawing, 1ml of 1M MgCl2 and 200 Kunitz units of DNAse were added. The solution was stirred at room temperature until the viscosity was reduced and then sonicated. The solution was then diluted and stirred with wash buffer to a final volume of 1L. After centrifugation (10000rpm, 20min), the pellet was washed with wash buffer and recovered again by centrifugation. The pellet was then dissolved in 60ml solubilization buffer and |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |