Refolding Record:
| Protein | |
|---|---|
| Protein Name | Single Chain Fv |
| Abbreviated Name | ScFv |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Western equine encephalitis |
| 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 | Das D, Kriangkum J, Nagata LP, Fulton RE, Suresh MR. (2004) J Virol Methods, 117, 169-177 |
| 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 | 12h |
| Expression Vector | pET22b+ |
| 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 | 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA |
| Solubilization Buffer | 6 M Urea |
| Refolding Buffer | see notes |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | nd |
| Refolding Time | 12-48h |
| Redox Agent | GSSG |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Set I: Refolding was done in buffer containing10mM DTT, 20 mM NaHCO3, 150 mM NaCl and 2 mM EDTA pH 8.0 (Sanchez et al., 1999) for 48 h. Finally, the folded protein was dialysed against phosphate buffer saline(pH 7.3). Set II: Solubilised denatured protein was treated with 1 mM GSH, 0.1 mM GSSG for overnight (16 h). Refolding was done in TA buffer (50 mMTris pH 8.0, 0.4 Ml-arginine) for 66 h. Final dialysis was done in 50 mM Tris pH 8.0 for 48 h. Set III: Solubilised denatured protein was treated with 5 mM GSH, 0.5 mM GSSG for overnight (16 h). Refolding was done in TA buffer (50 mMTris pH 8.0, 0.4 Ml-arginine) for 66h. Further dialysis was carried out in 50 mM Tris pH 8.0 for 48h. Set IV: Inclusion bodies were solubilised in 100 ml 2% sarcosyl, 50 mM Tris pH 10.0 by stirring at room temp for 2 h. Air oxidation was done by continued stirring of the sample in the presence of 50 M CuSO4 for 20 h. Urea was added to the sample to a final concentration of 6 M. Sarcosyl was removed from the sample by absorption twice with 10% Dowex 1X8 (w/v), stirring for 15 min each time (Kurucz et al., 1995). Solubilised denatured protein was adjusted to 100 g/ml and dialysed against TA buffer (50 mM Tris pH 8.0, 0.4 M -arginine) for 66 h. Finally, the sample was dialysed against 50 mM Tris pH 8.0 for 48 h. At the end of dialysis, any aggregation was removed by centrifugation at 10,000 rpm for 15 min at 4 °C. |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | nd |
| Notes | Various refolding protocols were used with solubilised inclusion bodies. Different reducing and oxidising environments such as dithiothreitol (DTT), air oxidation inpresence of CuSO4 and redox coupling such as glutathione(GSH, GSSG) along with different buffer systems were used to optimise the refolding conditions. |