Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cobrotoxin |
| Abbreviated Name | CBTX |
| SCOP Family | Snake venom toxins |
| Structure Notes | |
| Organism | Naja atra (Chinese cobra) |
| UniProt Accession | P60770 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 62 |
| Molecular Weight | 6956.6 |
| Pi | 8.5 |
| Molecular Weight | 6956.6 |
| Disulphides | 4 |
| Full Sequence |
LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hsieh HC, Kumar TK, Yu C. (2004) Biochem Biophys Res Commun., 320, 1374-1381 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal IgG tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | n/a |
| Expression Protocol | The vector map and amino acid sequence of CBTX are shown in Fig. 1. pCP expression vector containing cDNA sequence of fusion protein (IgG–CBTX) was transformed to BL21(DE3)pLysS for protein expression. An overnight culture of BL21(DE3)pLysS transformed cells was diluted 10 times in Luria–Ber- tani medium (AMRESCO, USA) supplemented with 100 mg/L ampicillin and 35 lg/mL chloroamphenicol (AMRESCO, USA) and incubated at 37 °C until OD600 reached 0.4. One millimolar of isopro-pylthio-B-D -galactoside (AMRESCO, USA) was then added and after 4 h of incubation, the cells were harvested by centrifugation at 6000 rpm for 20 min. Cell pellets from 1 L cultures were then resuspended in 30 mL resuspension buffer (150 mM NaCl, 25 mM phosphate buffer, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride (Sigma, USA). Cells were disrupted by French press at 1000 psi and the lysate was cleared by centrifugation. The expression of the target protein was checked by SDS–PAGE. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: affinity immobilization |
| Wash Buffer | 5 mM ammoniumacetate |
| Solubilization Buffer | n/a |
| Refolding Buffer | 0.1 M phosphate, 5 mM EDTA, 3 mM GSH, and 4 mM GSSG at pH 8.0 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 36 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3/4 |
| Refolding Protocol | Purification of recombinant CBTX. Clear cell lysate was applied to an IgG–Sepharose column (Amersham Biosciences, USA), then washed with 5 column volumes of phosphate buffer (150 mM NaCl, 25 mM phosphate buffer, pH 7.5) and followed by another 2 column volumes of 5 mM ammonium acetate at pH 5. After the refolding process, the hybrid protein was eluted with 0.5 M acetate at pH 3.4. Concentration of the IgG–CBTX fusion protein was estimated using the Bio-Rad protein assay kit. Refolding steps were introduced into the purification protocol after washing the column with 5 mM ammonium acetate. The IgG–Sepharose gel with the adsorbed fusion protein was washed with 2 column volumes of the renaturation buffer (0.1 M phosphate, 5 mM EDTA, 3 mM GSH, and 4 mM GSSG at pH 8.0). After incubation for 36 h at 4 °C, the purification was resumed at the ammonium acetate step. |
| Refolding Assay | NMR analysis,Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |