Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bcl-2 |
| Abbreviated Name | Bcl-2 |
| SCOP Family | Bcl-2 inhibitors of programmed cell death |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P10415 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 242 |
| Molecular Weight | 26265.9 |
| Pi | 6.75086 |
| Molecular Weight | 26265.9 |
| Disulphides | 0 |
| Full Sequence |
MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPA
ASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLH
LTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEY
LNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Anderson M, Blowers D, Hewitt N, Hedge P, Breeze A, Hampton I, Taylor I. (1999) Protein Expression and Purification, 15, 162-170 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| 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 | 5.5h |
| Expression Vector | pTB 375 |
| 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 | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris?HCl, 2.0 M urea, 2mM EDTA, 1 mM 2-mercaptoethanol, pH 8.0 |
| Solubilization Buffer | 50 mM Tris?HCl, 6.0M guanidine hydrochloride, 10 mM 2-mercaptoethanol, pH 8.0 |
| Refolding Buffer | 400 mM L-arginine, 10 mM Tris?HCl, 10 mM 2-mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 4mg/ml or less |
| Refolding Time | 20h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Solubilization buffer (50 ml) containing the dissolved inclusion bodies was thawed and, with rapid mixing, was added dropwise at a rate of 10 ml/min into 4.0 liters of refolding buffer (400 mM L-arginine, 10 mM Tris?HCl, 10 mM 2-mercaptoethanol, pH 8.0 at 4°C). The vessel containing the refolding mixture was made airtight and the refolding process allowed to continue with stirring for 20 h at 4°C. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 15% |
| Purity | 85% |
| Notes | |