Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single chain monoclonal antibody 4-4-20 |
| Abbreviated Name | SCA 4-4-20 |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | unknown |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | |
| Domain | None |
| Chimera | None |
| Variants | None |
| Chain Length | 219 |
| Molecular Weight | 24161.8 |
| Pi | 7.78 |
| Molecular Weight | 24161.8 |
| Disulphides | Unknown |
| Full Sequence | |
| Notes | None |
| Expression | |
|---|---|
| Report | Denzin LK, Whitlow M, Voss EW (1991) J Biol Chem, 266, 14095-14103 |
| Project Aim | Functional Studies |
| Fusion | N-terminal OmpA signal sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | GX6712 |
| Expression Temp | 42.0 |
| Expression Time | 1 |
| Expression Vector | pGX8772 |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris pH 8.0, 1mM EDTA, 0.1mM PMSF |
| Solubilization Buffer | 6M GdnHCl, 50mM TrisHCl pH 8.0, 10mM CaCl2, 100mM KCL, 0.1mM PMSF |
| Refolding Buffer | 50mM Tris pH 8.0, 10mM CaCl2, 50mM KCl, 0.1mM MPSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | |
| Refolding Protocol | 500ml of an overnight culture in 2xTY medium was used to inoculate 20 or 30L of media in a fermentor. Cells were grwon at 30degC until OD600 reached 1.0. Expression was induced by increasing the temperature to 42degC. Cells were incubated for a further 1h at 42degC, then harvested. The cell paste was suspended in 1ml of wash buffer per 3g of cell paste and passed through a French pressure cell at 20000psi twice. Following centrifugation (20000g, 45min), pellets were suspended in 1/2 the original volume of wash buffer, passed through the French press and centrifuged again (20000g, 30min). The pellets were then washed twic with wash buffer containing 0.5$ Triton X-100 followed one final wash without Triton X-100. The pellet was then solubilized in solubilization buffer and centrifuged (30min, 25000g. The protein was refolded by rapid dilutaion (1:200-1:1000) into refolding buffer. Following filtration and concentration, the refolded protein was purified by affinity or cation exchange chromatography |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 8-20% |
| Purity | |
| Notes | various light chain single point mutants also expressed, refolded and purified |