Refolding Record:
| Protein | |
|---|---|
| Protein Name | nitrophorins 1-4 from Rhodnius prolixus |
| Abbreviated Name | rNP1-4 |
| SCOP Family | Retinol binding protein-like |
| Structure Notes | |
| Organism | Rhodnius prolixus |
| UniProt Accession | x |
| 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 | 186 |
| Molecular Weight | 20482.9 |
| Pi | 6.34872 |
| Molecular Weight | 20482.9 |
| Disulphides | 2 |
| Full Sequence |
KCTKNALAQTGFNKDKYFNGDVWYVTDYLDLEPDDVPKRYCAALAAGTASGKLKEALYHYDPKTQDTFYDVSELQEESPG
KYTANFKKVEKNGNVKVDVTSGNYYTFTVMYADDSSALIHTCLHKGNKDLGDLYAVLNRNKDTNAGDKVKGAVTAASLKF
SDFISTKDNKCEYDNVSLKSLLTK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Andersen JF, Champagne DE, Weichsel A, Ribeiro JM, Balfour CA, Dress V, Montfort WR (1997) Biochemistry, 36, 4423-4428 |
| 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 | insoluble |
| 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,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. Two disulfide bonds are allowed to form AFTER folding. 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 |