Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bivalent e23 dsFv |
| Abbreviated Name | e23 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01756 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Bivalent e23 fused with PE38 |
| Variants | n/a |
| Chain Length | 811 |
| Molecular Weight | 87488.4 |
| Pi | 6.14 |
| Molecular Weight | 87488.4 |
| Disulphides | 2 |
| Full Sequence |
DVQLTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPWIYTTSNLASGVPARFSGGGSGTSYSLTVSRVEAEDAATYYCQQWSRSPPTFGGGSKEVQLQESGPEVVKPGGSMKISCKTSGYSFTGHTMNWVKQSHGKNLEWIGLINPYNGDTNYNQKFKGKATGTVDKSSSTAYMELLSLTSEDSAVYYCARRVTDWYFDVWGAGTTVTVSGGGGGSGGGGSGGGGSDVQLTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPWIYTTSNLASGVPARFSGGGSGTSYSLTVSRVEAEDAATYYCQQWSRSPPTFGGGSKEVQLQESGPEVVKPGGSMKISCKTSGYSFTGHTMNWVKQSHGKNLEWIGLINPYNGDTNYNQKFKGKATGTVDKSSSTAYMELLSLTSEDSAVYYCARRVTDWYFDVWGAGTTVTVSMLQGTKLMAEEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSQNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAFAANFPADSGDALLERNYPTGAEFLGDFFDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWRPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bera, T. K., Onda, M. Brinkmann, U., Pastan, I (1998) J Mol Biol, 281, 475-483 |
| Project Aim | Functional Studies |
| Fusion | None |
| 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 | 90min |
| Expression Vector | pYR40 |
| Expression Protocol | Cells were grown in superbroth containing 2% glucose, 0.05% MgSO4, and 100microg/ml ampicillin. After induction the periplasm was removed by osmotic shock. Spheroplasts were then resuspended in 50mM Tris, 20mM EDTA, pH 8.0 with 200microg/ml lysozyme and allowed to incubate for 60min at 20degC. Triton X-100 and NaCl were then added to the suspension to final concentrations of 2% an 0.5 M respectively and the mixture centrifuged (25,000g, 60min). Suspension and centrifugation was then repeated. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 3 |
| Cell Disruption Method | Osmotic shock |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 0.1 M Tris, pH 8.0 6 M guanidine, 2 mM EDTA, 0.3 M DTE |
| Refolding Buffer | 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM GSSG, 2 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSSG |
| Redox Agent Concentration | 8mM |
| Refolding Protocol | Inclusion bodies were solubilized in solubilization buffer and allowed to incubate for two hours at room temperature. Following incubation the suspension was centrifuged (30,000g, 30min) to remove insoluble particles. Renaturing was initated by a rapid 10 fold dilution with refolding buffer and was then allowed to incubate at 10degC. |
| Refolding Assay | Ligand Binding,Cytotoxicity Assays |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |