Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lymphotoxin-alpha also known as TNF-beta |
| Abbreviated Name | LT-alpha |
| SCOP Family | TNF-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01374 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 172 |
| Molecular Weight | 18661.2 |
| Pi | 8.94 |
| Molecular Weight | 18661.2 |
| Disulphides | Unknown |
| Full Sequence |
LPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jin H, Uddin MS, Huang YL, Teo WK. (1994) J Chem Technol Biotechnol., 59, 67-72 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | K12 HB101 |
| Expression Temp | 30.0 |
| Expression Time | 00 |
| Expression Vector | pCG402 |
| Expression Protocol | The E. coli K12 HBlOl strain cloned with rhTNF-P gene in plasmid pCG402 was obtained from the Institute of Molecular and Cell Biology, Singapore. Bacteria were grown in a 10 dm3 fermenter in production mediumI3 at 30°C under control of the trp promoter. Expression of the recombinant protein was induced by adding 50 mg dm-3 of IAA at mid-exponential phase at an optical density (OD,,,) of about 105. The amino acids proline and leucine were fed during fermentation |
| Method of Induction | 3 Beta-indoleacrylic acid (IAA) |
| Cell Density at Induction | OD 10.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Triton X-lOO/EDTA/lysozyme/PMSF |
| Solubilization Buffer | 8 mol dm-3 alkaline urea |
| Refolding Buffer | 30 mmol dm-3 Tris, 30 mmol dm-3 NaCI, 1 mmol dm-3 EDTA, pH 7 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | All procedures were performed at 4°C. The pool solution from the CM-Sepharose column was 30-fold diluted at a rate of 045cm3min-\' with renaturation buffer D containing 30 mmol dm-3 Tris, 30 mmol dm-3 NaCI, 1 mmol dm-3 EDTA, pH 7, and then concentrated to a final concentration of about 1 mg/m- in a stirred ultrafiltration cell (model 8200, 10000 MW cut-off, Amicon, Inc., USA) with an operating nitrogen pressure of 4 atm. |
| Refolding Assay | Cytotoxicity Assays |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |