Refolding Record:
| Protein | |
|---|---|
| Protein Name | CD23 antigen Also known as Low affinity immunoglobulin epsilon Fc receptor |
| Abbreviated Name | CD23 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P06734 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 173 |
| Molecular Weight | 19202.4 |
| Pi | 5.45 |
| Molecular Weight | 19202.4 |
| Disulphides | 4 |
| Full Sequence |
MELQVSSGFVCNTCPEKWINFQRKCYYFGKGTKQWVHARYACDDMEGQLVSIHSPEEQDFLTKHASHTGSWIGLRNLDLKGEFIWVDGSHVDYSNWAPGEPTSRSQGEDCVMMRGSGRWNDAFCDRKLGAWVCDRLATCTPPASEGSAESMGPDSRPDPDGRLPTPSAPLHS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Daniels BB, Askew SL, van de Venter M, Oosthuizen V. (2005) Cell Immunol., 234, 146-53 |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET23a |
| Expression Protocol | Recombinant CD23 was expressed in Escherichia coli BL21 (DE3) cells using 1 mM IPTG, which causes the bacterial cells to express the recombinant protein into inclusion bodies. The cells were harvested after 4 h induction at 37 °C, diluted in 50 mM Tris–HCl, pH 7.8, buffer, containing 0.3 M NaCl, and lysed by lysozyme (2 mg/mL) and sonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris–HCl, pH 7.8, buffer, containing 0.3 M NaCl, and lysed by lysozyme, 0.5% (w/v) lauryldimethylamine oxide |
| Solubilization Buffer | 20 mM Tris, 6 M guanidinium hydrochloride, 100 mM β-mercaptoethanol, pH 8, |
| Refolding Buffer | 0.1 M Tris, 0.15 M NaCl, 1.5 M arginine, 10 mM CaCl2, 5 mM reduced glutathione, and 0.5 mM oxidised glutathione, pH 8 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 72 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5/0.5 mM |
| Refolding Protocol | The cells were harvested after 4 h induction at 37 °C, diluted in 50 mM Tris–HCl, pH 7.8, buffer, containing 0.3 M NaCl, and lysed by lysozyme (2 mg/mL) and sonication. The inclusion bodies were washed at least 8 times with the latter buffer, containing 0.5% (w/v) lauryldimethylamine oxide to remove any contaminating E. coli proteins. The washed inclusion bodies were dissolved (1:10, v/v) in 20 mM Tris, 6 M guanidinium hydrochloride, 100 mM β-mercaptoethanol, pH 8, and the recombinant protein was renatured using rapid dilution into 0.1 M Tris, 0.15 M NaCl, 1.5 M arginine, 10 mM CaCl2, 5 mM reduced glutathione, and 0.5 mM oxidised glutathione, pH 8 at 4 °C. Following concentration by ultrafiltration, the refolded protein was purified using a Superdex 75 gel filtration chromatography column equilibrated and developed with 0.1 M Tris, 0.15 M NaCl, and 10 mM CaCl2, pH 8. The level of contaminating lipopolysaccharide (LPS) in the purified protein was determined using the Limulus Amebocyte Lysate Assay (Charles River Endosafe, Charleston, USA), and the final concentration of LPS (<100 ng/mg protein) added to cell cultures was never more than 10 pg/mL. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 1.5 M |
| Refolding Yield | 0.5-3 mg/L cul. |
| Purity | n/a |
| Notes | n/a |