Refolding Record:
| Protein | |
|---|---|
| Protein Name | Serum Retinol Binding Protein |
| Abbreviated Name | RBP |
| SCOP Family | Retinol binding protein-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P02753 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 204 |
| Molecular Weight | 23010.0 |
| Pi | 5.76211 |
| Molecular Weight | 23010.0 |
| Disulphides | 3 |
| Full Sequence |
MKWVWALLLLAALGSGRAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIV
AEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGND
DHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLA
RQYRLIVHNGYCDGRSERNLL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Xie Y, Lashuel HA, Miroy GJ, Dikler S, Kelly JW. (1998) Protein Expression and Purification, 14, 31-37 |
| Project Aim | Undefined |
| 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 | 8h |
| Expression Vector | unknown |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 25mM Tris-HCl, 5M guanidinium chloride, 10mM DTT, pH 9.0 |
| Refolding Buffer | 25mM Tris-HCl, 0.3mM cystine, 3mM cysteine, 1mM EDTA, 10-fold molar excess Vitamin A, 100 micromolar retinol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.25mg/ml |
| Refolding Time | 5h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The oxidative refolding of RBP is carried out by dilution of the denatured and reduced stock solution of RBP (1.25 mg/mL) into a redox refolding buffer containing a 10-fold molar excess of vitamin A, yielding a final RBP concentration of 0.25 mg/mL. To initiate refolding, 1 vol (typically 100 mL) of ;1.25 mg/mL denatured and reduced RBP in 5.0 M GdmCl is rapidly mixed with 4 vol of the redox refolding buffer containing vitamin A, while magnetic stirring the solution vigorously (final [GdmCl] 5 1.0 M). The refolding solution should be kept in an ice bath or cold room for 5 h after refolding is initiated. Refolding RBP in the absence of vitamin A affords a poor yield of properly folded RBP (20%). The redox refolding buffer is composed of 0.3mM cystine, 3 mM cysteine, 1 mM EDTA, 25 mM Tris-HCl buffer (pH 9.0), and is degassed by nitrogen sparging for 20 min. After degassing, 3.2 mL of 15 mM retinol (dissolved in ethanol under argon) is added to the redox diluting buffer right before adding denatured RBP. To avoid aggregation, all solutions were prechilled in an ice bath or in cold room before combining the refolding buffer with denatured RBP at 4°C |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 50-60% |
| Purity | 50% |
| Notes | |