Refolding Record:
| Protein | |
|---|---|
| Protein Name | Nitrophorin from Cimex lectularius |
| Abbreviated Name | cNP |
| SCOP Family | Inositol polyphosphate 5-phosphatase (IPP5) |
| Structure Notes | |
| Organism | Cimex lectularius |
| UniProt Accession | O76745 |
| 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 | 307 |
| Molecular Weight | 33624.0 |
| Pi | 7.65609 |
| Molecular Weight | 33624.0 |
| Disulphides | 0 |
| Full Sequence |
MKLLLSAGAALAFVLGLCAAGSPPAQLSVHTVSWNSGHERAPTNLEELLGLNSGETPDVI
AVAVQGFGFQTDKPQQGPACVKNFQSLLTSKGYTKLKNTITETMGLTVYCLEKHLDQNTL
KNETIIVTVDDQKKSGGIVTSFTIYNKRFSFTTSRMSDEDVTSTNTKYAYDTRLDYSKKD
DPSDFLFWIGDLNVRVETNATHAKSLVDQNNIDGLMAFDQLKKAKEQKLFDGWTEPQVTF
KPTYKFKPNTDEYDLSATPSWTDRALYKSGTGKTIQPLSYNSLTNYKQTEHRPVLAKFRV
TL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Weichsel, A. et al (2005) Proc. Natl. Acad. Sci. USA, 102, 594-599 |
| Project Aim | Structure-Function |
| 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 | 3 hr |
| Expression Vector | pET17b |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 30 mM Tris, 20 mM NaCl |
| Solubilization Buffer | 6M Guanidine HCl, 5mM DTT, 30mM Tris, pH7.5, 1mM EDTA |
| Refolding Buffer | 30 mM Tris, pH 7.5, 800mM NaCl, 10mM DTT, 15 uM PMSF, 1mM EDTA |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | dilute |
| Refolding Time | 12 hr |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Briefly: Inclusion bodies are washed and solubilized in guanidine HCL. This material is dripped into a renaturation buffer with HIGH SALT (800 mM, 50-fold dilution). The protein is allowed to refold overnight at 4 deg. Dialysis to remove salt and guanidine. Heme is titrated in after this, generally, but can be added after concentration and purification if necessary (e.g. using a synthetic analogue). A Q-sepharose column (anion exchange) removes excess heme, and sephacryl column (size exclusion) removes a contaminant. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | ~50% |
| Purity | ~95% |
| Notes | Full length protein without secretion tag (normally exported from the cell in the insect). |