Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single Chain Fv |
| Abbreviated Name | ScFv |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Clostridium thermocellum |
| 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 | Berdichevsky Y, Lamed R, Frenkel D, Gophna U, Bayer EA, Yaron S, Shoham Y, Benhar I. (1999) Protein Expression and Purification, 17, 249-259 |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pFEKCA3d |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50 mM Tris-HCl (pH 8.0), 20 mM EDTA |
| Solubilization Buffer | 8M Urea, 50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 50 mM DTE |
| Refolding Buffer | 20 mMTris(HCl), pH 7.5,1 mM EDTA, 250 mM NaCl (TBS) which was adjusted to pH 9.5 by addition of NaOH |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 9.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 24-72h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Refolding was carried out by rapid dilution of the solubilized and reduced inclusion bodies into 100 volumes of 20 mMTris(HCl), pH 7.5/1 mM EDTA/250 mM NaCl (TBS) which was adjusted to pH 9.5 by addition of NaOH. 3.3 mg of inclusion bodies protein was diluted into 100 ml of TBS to a final protein concentration of33 |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 1.0mg |
| Purity | nd |
| Notes | |