Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cardiotoxin analogue III |
| Abbreviated Name | CTX III |
| SCOP Family | Snake venom toxins |
| Structure Notes | |
| Organism | Taiwan cobra |
| UniProt Accession | P60301 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 82 |
| Molecular Weight | 9038.2 |
| Pi | 9.26424 |
| Molecular Weight | 9038.2 |
| Disulphides | 4 |
| Full Sequence |
MKTLLLTLVVVTIVCLDLGYTLKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCI
DVCPKSSLLVKYVCCNTDRCN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kumar TK, Yang PW, Lin SH, Wu CY, Lei B, Lo SJ, Tu SC, Yu C (1996) Biochemical and Biophysical Research Com, 219, 450-456 |
| 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 | 5h |
| Expression Vector | pET21b |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Reverse phase chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | unknown |
| Solubilization Buffer | 0.1M Tris-HCl, 2% betamercaptoethanol, 8M urea, pH 8.7 |
| Refolding Buffer | 0.1M Tris-HCl, 10mM GSSG |
| Pre-Refolding Purification | Reverse phase chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 36h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet on centrifugation was dissolved in 0.1 M Tris-HCl (pH 8.7) containing 2% b-mercaptoethanol and 8 M urea at room temperature. The process of dissolution of the pellet was allowed to continue for at least 3 h. All HPLC runs were carried out on a semi-preparative reverse phase C18 m-Bondapak column using 0?0% gradient of acetonitrile containing 0.1% trifluoro acetic acid over a time period of 60 minutes. The HPLC runs were carried out on a Hitachi machine (Model L-400). The eluted proteins were detected using 280 nm absorbance. The samples obtained from HPLC runs were dried using a speed-vac (Savant Co., USA). The dried powder comprising the denatured or partially structured CTX III was redissolved in 0.1 M Tris-HCl (pH 8.7) containing 8 M urea and 0.15 M b-mercaptoethanol and subjected to slow refolding against 0.1 M Tris-HCl (pH 8.0) containing 10 mM oxidized glutathione for about 36 h. |
| Refolding Assay | 1H chemical shift (ppm) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 40mg/L culture |
| Purity | |
| Notes | |