Refolding Record:
| Protein | |
|---|---|
| Protein Name | Low affinity IgE Fc Receptor |
| Abbreviated Name | IgE Fc Receptor |
| SCOP Family | C-type lectin domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P06734 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | 16 kDa fragment of lectin homology domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 326 |
| Molecular Weight | 36468.8 |
| Pi | 5.38303 |
| Molecular Weight | 36468.8 |
| Disulphides | 4 |
| Full Sequence |
MEEGQYSEIEELPRRRCCRRGTQIVLLGLVTAALWAGLLTLLLLWHWDTTQSLKQLEERA
ARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQADL
SSFKSQELNERNEASDLLERLREEVTKLRMELQVSSGFVCNTCPEKWINFQRKCYYFGKG
TKQWVHARYACDDMEGQLVSIHSPEEQDFLTKHASHTGSWIGLRNLDLKGEFIWVDGSHV
DYSNWAPGEPTSRSQGEDCVMMRGSGRWNDAFCDRKLGAWVCDRLATCTPPASEGSAESM
GPDSRPDPDGRLPTPSAPLHS
|
| Notes | NMR solution of 16 kDa fragment of lectin homology domain. |
| Expression | |
|---|---|
| Report | Daniels BB, Askew SL, van de Venter M, Oosthuizen V. (2005) Cell Immunol., 234, 146-53 |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 4 hrs |
| Expression Vector | pET23a |
| Expression Protocol | Not stated |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 0.3M NaCl, 0.5% LDAO, 0.2% sodium azide, pH 7.8 |
| Solubilization Buffer | 20mM Tris-HCl, 6M guanidinium HCl, 100mM b-mercaptoethanol, 0.02% sodium azide, pH 8.0 |
| Refolding Buffer | 100mM Tris-HCl, 150mM NaCl, 1.5M arginine, 10mM CaCl2, 5mM GSH, 0.5mM GSSG, pH 8.0 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 100 µg/mL |
| Refolding Time | 72 hours |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1.5 M |
| Refolding Protocol | E. coli ROSETTA or BL21 (DE3) cells were grown in 2L LB medium, containing 100µg/mL ampicillin and 36 µg/mL chloramphenicol (for ROSETTA cells only) until reaching mid-log phase (OD600nm ~ 0.6). Expression was induced with 1mM IPTG and growth continued at 37ºC for 4hours. Cells were harvested by centrifugation at 4ºC and 5000xg, before lysis with lysozyme (2mg/mL) and sonication. Inclusion bodies were washed with wash buffer at least 8 times to remove E. coli proteins. The inclusion bodies were solublised in solublization buffer prior to refolding. Solublised inclusion bodies (~50mg protein) were added dropwise into 500mL refolding buffer, over ~16 hours, with stirring. The refold was left for 72 hours to complete refolding. Protein was concentrated by ultrafiltration and purified by gel filtration on Superdex75, using PBS, pH 7.4 as buffer. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | n/a |
| Refolding Yield | 20% |
| Purity | Homogen |
| Notes | n/a |