Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single Chain Fv |
| Abbreviated Name | ScFv |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | Q9HCC1 |
| 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 | 113 |
| Molecular Weight | 12242.6 |
| Pi | 8.674 |
| Molecular Weight | 12242.6 |
| Disulphides | 4 |
| Full Sequence |
EVQLVESGGGVVRPGGSLRISCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY
ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRRYALDYWGQGTLV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Umetsu M, Tsumoto K, Hara M, Ashish K, Goda S, Adschiri T, Kumagai I. (2003) J Biol Chem, 278, 8979-8987 |
| Project Aim | Structure-Function |
| 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 | Not stated |
| Expression Vector | pUT7 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl (pH 8.0) buffer with 4% Trition X-100 and 200mM NaCl |
| Solubilization Buffer | 50mM Tris-HCl (pH 8.0) buffer with 6M GdnHCl and 200mM NaCl |
| Refolding Buffer | 50mM Tris-HCl (pH 8.0) with 6M GdnHCl, 200mM NaCl and 1mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The concentration of the unfolded scFv was adjusted to 7.5 µM in the 50 mM Tris-HCl (pH 8.0) buffer with 6 M GdnHCl, 200 mM NaCl, and 1 mM EDTA, and the fragment was reduced by the addition of 2-mercaptoethanol (-ME) at a 50-fold molar excess relative to the protein. After -ME was removed by dialysis against the same Tris-HCl buffer without -ME, the unfolded scFv was refolded by gradual removal of GdnHCl by means of stepwise dialysis from 6 to 0 M through 3, 2, 1, and 0.5 M by one of the following four methods; (a) the concentration of GdnHCl was decreased by stepwise dialysis without any additives; (b) GSSG (50-fold molar excess relative to protein) was added at the 1 and 0.5 M GdnHCl stages; (c) 400 mM L-arginine was added at the 1 and 0.5 M GdnHCl stages; and (d) GSSG (50-fold molar excess relative to protein) and L-arginine (400 mM) were added at the 1 and 0.5 M GdnHCl stages. |
| Refolding Assay | Fluorescence |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | Not Stated |
| Notes | Absorption, CD, Fluorescence, and FT-IR Spectroscopy experiments were also carried out |