Refolding Record:
| Protein | |
|---|---|
| Protein Name | translation initiation factor eIF-4E |
| Abbreviated Name | eIF-4E |
| SCOP Family | Translation initiation factor eIF4e |
| Structure Notes | |
| Organism | Xenopus laevis |
| UniProt Accession | P48597 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 216 |
| Molecular Weight | 24634.7 |
| Pi | 5.78881 |
| Molecular Weight | 24634.7 |
| Disulphides | 0 |
| Full Sequence |
MAAVEPENTNPQSTEEEKETGQEIVSPDQYIKHPLQNRWALWFFKNDKSKTWQANLRLIS
KFDTVEDFWALYNHIQLSSNLMSGCDYSLFKDGIEPMWEDEKNKRGGRWLITLNKQQRRN
DLDRFWLETLMCLIGESFDEHSDDVCGAVVNVRAKGDKIAIWTTEFENKDAVTHIGRVYK
ERLGLPAKVVIGYQSHADTATKSGSTTKNRFVV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Wakiyama, M. Sakai, N. Kojima, S. & Miura, K. (1997) FEBS Letters, 409, 407-410 |
| Project Aim | Structure-Function |
| 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 | 16h |
| Expression Vector | pET11d |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Column Refolding Combination |
| Wash Buffer | None |
| Solubilization Buffer | 20mM HEPES/KOH, pH7.5 20mM DTT, 1mM EDTA, 6M Guanidine hydrochloride |
| Refolding Buffer | 20mM HEPES/KOH, pH 7.5, 1mM EDTA, 3mM glutathione, 0.3mM glutathione disulfide, 1.5M KOAc |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 2mg per 10ml refolding buffer |
| Refolding Time | 12 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet containing 2 mg of eIF-4E protein was dissolved in 10 ml of solution A (20 mM HEPES/KOH, pH 7.5, 20 mM dithiothreitol, 1 mM EDTA, 6 M guanidine-hydrochloride) and incubated on ice for 2 h with occasionally shaking. The protein solution was diluted with 100 ml of solution B (20 mM HEPES/KOH, pH 7.5, 1 mM EDTA, 3 mM glutathione, 0.3 mM glutathione disulfide, 1.5 M KOAc) and incubated at 4°C for 12 h. Then the protein solution was diluted with solution C (20 mM HEPES/KOH, pH 7.5, 1 mM EDTA) to achieve a final concentration of 150 mM KOAc and applied to a 0.5 ml 7-methyl-GTP-Sepharose 4B column. The column was washed with 50 ml of solution D (20 mM HEPES/KOH, pH 7.5, 1 mM EDTA, 100 mM KCl). The refolded eIF-4E was eluted with 0.1 mM m7GTP in solution D and concentrated by Centriprep-30 (Amicon). |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |