Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta-bungarotoxin A5 chain |
| Abbreviated Name | Beta-Bgt A5 |
| SCOP Family | Small Kunitz-type inhibitors & BPTI-like toxins |
| Structure Notes | |
| Organism | Bungarus multicinctus (Many-banded krait) |
| UniProt Accession | P59018 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 121 |
| Molecular Weight | 13711.5 |
| Pi | 7.91308 |
| Molecular Weight | 13711.5 |
| Disulphides | 6 |
| Full Sequence |
MNLY QFKEMIRYTI PCEKTWGEYA DYGCYCGAGG SGRPIDALDR CCYVHDNCYG DAEKKHKCNP KTQSYSYKLT KRTIICYGAA GTCGRIVCDC DRTTALCFGN SEYIEGHKNI DTARFCQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chang L, Wu P, Chang C (1996) Biochemical and Biophysical Research Com, 221, 328-332 |
| Project Aim | Recombinant Protein Expression |
| 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 | 4h |
| Expression Vector | pT7-7 |
| Expression Protocol | Cells were grow in LB medium containing 50microg/ml ampicillin at 37degC. When OD550 reached 1.0, 1mM IPTG was added and cells were grown for a further 4h. Cells were harvested and lysed by sonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 550 = 1.0 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 1:8M urea 2:6M GdnHCl |
| Refolding Buffer | 0.035M Tris-acetate pH 8.0, 0.9M GdnHCl, 0.5M EDTA, 3mM cysteine |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.2mg/ml |
| Refolding Time | 1 week |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 3mM,3mM |
| Refolding Protocol | Following sonication, the insoluble pellet was resuspended in 8M urea solution by vigorous stirring and intermittent sonication over a 30min period. The mixture was centrifuged (30min, 27000g) and the supernatant was dialyzed against three changes of 4L dH2O and subjected to lyophilization. 10mg lyophilized proteins was dissolved in 1ml 6M GdnHCl and reduced with 2% 2-mercaptoethanol. The reaction was allowed to proceed at 37degC for 24h, then the solution was desalted by passage through a Sephadex G-25 column equilibrated with 0.6% acetic acid containing 6M GdnHCl. The protein was diluted with Tris buffer containing 0.6M GdnHCl to a final protein concentration of 0.2mg/ml, then the protein was reoxidized at room temperautre in refolding buffer. The refolding reaction was allowed to proceed for one week, the protein was then further purified by HPLC |
| Refolding Assay | Enzyme activity,Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |