Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-13 receptor alpha-2 |
| Abbreviated Name | IL-13R alpha 2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Dog (Canis familiaris) |
| UniProt Accession | Q95LF0 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 317 |
| Molecular Weight | 37134.7 |
| Pi | 4.71 |
| Molecular Weight | 37134.7 |
| Disulphides | Unknown |
| Full Sequence |
SMLSNAEIK VNPPQDFEIV DPGYLGYLSL QWQPPLFPDN FKECTIEYEL KYRNIDSENW KTIITKNLHY KDGFDLNKGI EAKINTLLPA QCTNGSEVRS SWAETTYWTS PQGNRETKIQ DMDCVYYNWQ YLVCSWKPGM GVHFDTNYQL FYWYEGLDHS AECTDYIKVN GKNMGCRFPY LESSDYKDFY ICVNGSSESQ PIRPSYFIFQ LQNIVKPMPP DYLSLTVKNS EEINLKWNMP KGPIPAKCFI YEIEFTEDGT TWVTTTVENE IQITRTSNES QKLCFLVRSK VNIYCSDDGI WSEWSDEQCW KGDIWKET
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Tang L, Morales T, Boroughs KL, Cailles Lo-Keiser K, Sellins K, Stedman K, McCall C, McDermott MJ. (2003) Molecular Immunology, 39, 719-727 |
| 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 | 30.0 |
| Expression Time | not stated |
| Expression Vector | p lambda cro |
| Expression Protocol | Cells were grown in LB broth at 30degC until optical density reached 15-20. |
| Method of Induction | Autoinduction |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 25mM Tris-HCl, 10mM EDTA, 0.5% deoxycholate, pH 7.5 |
| Solubilization Buffer | 25 mM Tris, pH 9.5, 8 M urea, 50 mM betamercaptoethanol |
| Refolding Buffer | 0.6 M urea, 45 mM Tris, 1.0 mM EDTA, 2.5 mM oxidized glutathione (GSSG) pH 10.0 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 10.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.08-0.1mg/ml |
| Refolding Time | 12-16h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 2.5mM |
| Refolding Protocol | Soluble rcaIL-13R2 (25 g cells in 250 ml of solubilization buffer) was loaded onto a 20 ml DEAE Sepharose column, previously equilibrated with 25 mM Tris, pH 9.0, 8 M urea, 10 mM DTT. Bound protein was eluted at a flow rate of 5.0 ml/min with 25 mM Tris, pH 9.0, 8 M urea, 10 mM DTT, 1 M NaCl (DEAE elution buffer) using a linear gradient to 0.7 M NaCl over 15 column volumes (CV). The receptor recovered in the DEAE pool (80-90 mg protein at 0.8 mg/ml) was reduced by the addition of 6 mM DTT, 0.05% Tween 20 and incubated at 30degC for 2 h. Refolding was initiated by dilution of the reaction mixture with 50 mM Tris, pH 10.0 to yield a refolding reaction mixture containing a final protein concentration of 80-90 microg/ml, 0.6 M urea, 45 mM Tris, pH 10.0, 1.0 mM EDTA, and 2.5 mM oxidized glutathione (GSSG). Refolding was performed at 4degC overnight with gentle stirring. The refolding mixture was dialyzed extensively against phosphate buffered saline (PBS), pH 7.2, at 4degC and clarified by centrifugation at 10,000g for 30 min. The purified, refolded rcaIL-13R2 was fractionated by 12% SDS-PAGE under reducing conditions, and then transferred onto a ProBlott membrane (Applied Biosytems, Foster City, CA) following manufacturer’s protocol. The dominant protein band on the membrane with predicted molecular mass for rcaIL-13R2 was removed for N-terminal amino acid sequencing. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |