Refolding Record:
| Protein | |
|---|---|
| Protein Name | Voltage-dependent Anion Channel 1 |
| Abbreviated Name | rhVDAC1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P21796 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 292 |
| Molecular Weight | 31895.8 |
| Pi | 8.81948 |
| Molecular Weight | 31895.8 |
| Disulphides | 0 |
| Full Sequence |
MRGSHHHHHH AVPPTYADLG KSARDVFTKG YGFGLIKLDL KTKSENGLEF TSSGSANTET TKVTGSLETK YRWTEYGLTF TEKWNTDNTL GTEITVEDQL ARGLKLTFDS SFSPNTGKKN AKIKTGYKRE HINLGCDMDF DIAGPSIRGA LVLGYEGWLA GYQMNFETAK SRVTQSNFAV GYKTDEFQLH TNVNDGTEFG GSIYQKVNKK LETAVNLAWT AGNSNTRFGI AAKYQIDPDA CFSAKVNNSS LIGLGYTQTL KPGIKLTLSA LLDGKNVNAG GHKLGLGLEF QA
|
| Notes | Other constructs also refolded: aa 1-100, 1-145, 1-196, 1-245, 12-283, 24-283, 127-283, 176-283 |
| Expression | |
|---|---|
| Report | Shi Y, Jiang C, Chen Q, Tang H (2003) Biochemical and Biophysical Research Com, 303, 475-482 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XL1-Blue |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pQE30a |
| Expression Protocol | The protein was expressed by incubation at 37degC for 4h with 1mM IPTG. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Cobalt-chelating chromatography |
| Wash Buffer | mixtures of solubilization and refolding buffers (different ratios) + 10mM imidazole |
| Solubilization Buffer | 4M GdnHCl, 20mM TrisHCl pH 7.9, 500mM NaCl, 10% glycerol |
| Refolding Buffer | 2% OG, 20mM Tris pH 7.9, 500mM NaCl, 10% glycerol |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.9 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Pelleted cells were resuspended in buffer A(20 mM Tris, pH 7.9, 200mM NaCl and 1mM PMSF) and sonicated and centrifuged. The inclusion bodies were washed extensively in buffer A then solubilized in solubilization buffer for 30 min at 4degC with gentle stirring. The cell lysate was then centrifuged and the supernatant was collected and loaded onto Talon beads equilibrated with solubilization buffer. The Talon beads were washed with 5 volumes of solubilization buffer, followed by three volumes of solubilization buffer mixed with refolding buffer at ratios of 1:3, 1:7 and 1:15 (solubilization:refolding) with 10mM imidazole. The beads were then washed with a further 10 column volumes of refolding buffer with 10mM imidazole, then the refolded protein was eluted with 4 column volumes of refolding buffer with 100mM imidazole. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | 20mg/L |
| Purity | |
| Notes | |