Refolding Record:
| Protein | |
|---|---|
| Protein Name | scFv mAbB23, specific for apolipoprotein B-100 |
| Abbreviated Name | scFv mAbB23 |
| 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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 245 |
| Molecular Weight | 25728.6 |
| Pi | 5.51 |
| Molecular Weight | 25728.6 |
| Disulphides | 0 |
| Full Sequence |
MQAVVTQESAL TTSPGETVTL TCRSNTGAVT TSNYASWVQE KPDHLFTGLI GGTNNRVPGV PARFSGSLIG DKAALTITGA QTEDEAIYFC ALWNSNHWVF GGGTKLTVLG GGGGSGGGGSGGGGS EVQLVESGPG LVAPSQSLSI TCTVSGFSLT DYGVSWIRQP PGKGLEWLGV IWAGGSTFYN SALKSRLSIN KDNSKSQVFL KMNSLHTDDT AMYYCVKHED RYDWYFDVWG AGTTVTVSS
|
| Notes | V-light chain - linker - V-heavy chain |
| Expression | |
|---|---|
| Report | Lee MH, Park TI, Park YB, Kwak JW. (2002) Protein Expression and Purification, 25, 166-173 |
| 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 | BLR(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET29b |
| Expression Protocol | Cells were grown at 37degC in LB media with 50 micrograms/ml kanamycin. When OD600 reached 0.6, expression was induced by the addition of 0.1mM IPTG and further incubation for another 4h. Cells were harvested by centrifugation and then resuspended in wash buffer. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM TrisHCl, 100mM KCl, 1mM EDTA, 0.1mM PMSF pH 8.0 |
| Solubilization Buffer | 6M GdnHCl, 50mM TrisHCl, 10mM CaCl2, 50mM KCl pH 8.0 |
| Refolding Buffer | 50mM TrisHCl, 10mM CaCl2, 50mM KCl, 0.1mM PMSF pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 20.0 |
| Protein Concentration | 3.0 mg/ml |
| Refolding Time | >20h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were sonicated, then centrifuged (15min x 12000g). The pellet was then resuspended in wash buffer and sonicated again, centrifuged and resupsended in wash buffer twice more. The inclusion bodies were then dissolved in solubilization buffer and centrifuged (15min x 12000g). The supernatant was incubated at room temperature for 2h to allow complete solubilization to occur. The protein was then folded by a gradual 10-fold dilution of the protein into the refolding buffer, the refolding buffer was added slowly (50 microlitres/min) with gentle mixing. The mixture was then left to stand at 4degC for 20h to allow the solubilized inclusion bodies to refold. The mixture was then dialyzed against refolding buffer to remove any remaining GdnHCl. The refolded protein was then loaded onto a prepared apoB-100-coupled affinity matrix column equilibrated with 50mM TrisHCl pH 8.0. After washing seeral times with the Tris buffer, the bound protein was eluted with 0.1M glycine (pH 3.0) and the eluted protein was immediately neutralized with 1M TrisHCl (pH 8.0). The eluate was then dialyzed against PBS and inf necessary concentrated with ammonium sulfate to a final 65% saturation. |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 15-20 mg/ml |
| Purity | |
| Notes | Reducing agents were added but did not enhance the refolding yield of scFv. |