Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single chain Fv HyHEL-10 |
| Abbreviated Name | scFv HyHEL-10 |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | n/a |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | |
| Domain | None |
| Chimera | None |
| Variants | None |
| Chain Length | 0 |
| Molecular Weight | 38679.9 |
| Pi | 0.0 |
| Molecular Weight | 38679.9 |
| Disulphides | 0 |
| Full Sequence | |
| Notes | None |
| Expression | |
|---|---|
| Report | Tsumoto, K, Shinoki, K, Kondo, H, Uchikawa M, Juji T, Kumagai, I (1998) J Immunol Methods, 219, 119-129 |
| 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 | n/a |
| Expression Vector | pUT7 |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 50mM Tris pH 7.6, 6M GdnHCl, 10mM 2-mercaptoethanol |
| Refolding Buffer | 100mM TrisHCl (pH 8.0), 200mM NaCl |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | days |
| Redox Agent | Beta-mercaptoethanol/Cystamine |
| Redox Agent Concentration | |
| Refolding Protocol | Cells were grown in LB broth at 28degC, expression was induced by the addition of 1mM IPTG followed by incubation at 37degC. The cells were harvested and suspended in phosphate-buffered saline (PBS). Following sonication, the solution was centrifuged at 12000rpm for 60min at 4degC. The pellet was then solubilized in solubilization buffer overnight at 4degC. The mixture was then centrifuged (12000rpm) and the supernatant was subjected to a Sephacryl S-200 column in 100mM TrisHCl (pH 8.0), 6M GdnHCl, 200mM NaCl. The purified protein was then reduced prior to refolding by incubation with 100mM TrisHCl (pH 8.0), 6M GdnHCl, 200mM NaCl and 10mM 2-mercaptoethanol for at least 30min at room temperature. The 2-mercaptoethanol was then removed by dialysis against 100mM TrisHCl (pH 8.0), 6M GdnHCl, 200mM NaCl. GdnHCl was then diluted by dialysis against refolding buffer containing 3M GdnHCl, followed by further step-wise dialyses against refolding buffer containing decreasing concentrations of GdnHCl (2,1,0.5,0M). Each dialysis step was performed overnight. A mixed disulfide derivative of the reduced and denatured inclusion bodies was formed by dialysis against 100mM TrisHCl (pH 8.0), 200mM NaCl, 0.375mM oxidized glutathione (50x molar excess of refolding protein) at certain GdnHCl concentrations (3-0.5M). In some experiments 2-mercaptoethanol (50x molar excess of protein) was added to the mixture at certain GdnHCl stages. The refolded protein was centrifuged (10000rpm x 15min) and the supernatant was loaded onto a HEL-sepharose column, comprising about 1-mg HEL/ml gel bound to CNBr-activated-Sepharose 4B, which had been preequilibrated with PBS. The column was washed with PBS, then 100mM TrisHCl (pH 8.5) with 500mM NaCl, then the protein was eluted with 100mM glycine pH 2.0, then the eluate was rapidly neutralized with 1M TrisHCl pH 7.5. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 150mg/L |
| Purity | |
| Notes | - same expression and purification protocol applicable for D10 scFv, up to HEL-sepharose step. - Multi-step dialysis more productive than single-step dialysis - best yield of protein when GSSG added at 1M GdnHCl dialysis step - addition of 400mM L-arginine in refolding buffer at 0.5M GdnHCl dialysis step increased yield - see also Umetsu et al. (2003) J Biol Chem 278:8979-8987 for further analysis of effects of L-arginine and GSSG on refolding |