Refolding Record:
| Protein | |
|---|---|
| Protein Name | scFv anti-crotoxin |
| Abbreviated Name | scFV anti-crotoxin |
| 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 | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 223 |
| Molecular Weight | 24343.1 |
| Pi | 5.46912 |
| Molecular Weight | 24343.1 |
| Disulphides | Unknown |
| Full Sequence |
maqvklqqsg aelvrsgasv klsctasgfn ikdyymnwvk qrpeqglewi gwidpengdt eyapkfqgka tmtadtssnt aylqlsslts edtavyycnv gyygssslfd ywgq dieltqsptt maaspgekit itcsanssis snylhwyqqk pgfspklliy rtsnlasgvp arfsgsgsgt sysltigtme aedvatyycq lgnsiprtfg ggtkleikr
|
| Notes | See GenBank accession numbers U14531 and U14532 for V(h) and V(l) chains |
| Expression | |
|---|---|
| Report | Meng J, John TR, Kaiser II (1995) Biochemical Pharmacology, 50, 1969-1977 |
| Project Aim | Drug 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 | 3h |
| Expression Vector | pET11b |
| Expression Protocol | Cells were grown at 37degC in 250ml LB medium until A600 reached 0.5, at which point expression was induced with 2mM IPTG. Cells were grown a further 3h, then harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM TrisHCl pH 8.0, 2mM EDTA |
| Solubilization Buffer | 50mM TrisHCl pH 8.0, 7.5M GdnHCl, 3mM EDTA |
| Refolding Buffer | phosphate buffered saline (PBS) pH 7.4 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet was washed twice by resuspension in 25ml wash buffer and centrifugation (12000g, 10min). The pellet was then dissolved in 5ml solubilization buffer. Refolding was performed by dilution into 1L of refolding buffer, the solution was left to stand at room temperature for 48h, then the solution was concentrated and the protein purified using a crotoxin affinity column. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 50% recovery |
| Purity | 95% |
| Notes | |