Refolding Record:
| Protein | |
|---|---|
| Protein Name | High affinity immunoglobulin epsilon alpha chain |
| Abbreviated Name | FcEpsilonRI-alpha |
| SCOP Family | I set domains |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P12319 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain aa.1-172 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 173 |
| Molecular Weight | 20063.4 |
| Pi | 4.97298 |
| Molecular Weight | 20063.4 |
| Disulphides | 2 |
| Full Sequence |
MVPQKP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE SEPVYLEVFS DWLLLQASAE VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE ALKYWYENHN ISITNATVED SGTYYCTGKV WQLDYESEPL NITVIKA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Takai T, Okumura K, Ra C. (2001) Biosci Biotechnol Biochem., 65, 79-85 |
| Project Aim | Functional Studies |
| 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 | 4 |
| Expression Vector | pGEMEX |
| Expression Protocol | 170ml of LB media was inoculated with 1.7ml of an overnight LB culture of cells, with 50 micrograms/ml ampicillin. After 2.5h incubation at 30degC, expression was induced with 1mM IPTG. After a further 4h incubation, the cells were harvested by centrifugation and resuspended in 6ml x 100mM TrisHCl, 10mM EDTA, pH 7.5. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6Murea, 20mM TrisHCl, pH 8.5 |
| Refolding Buffer | 20mM Tris, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 12h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Following sonication, the lysate was centrifuged (15min x 7000g) and the inclusion bodies were then solubilized in 50ml solubilization buffer. The protein was then dialyzed againse refolding buffer for 12h. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | Same protocol as per Der f2 - see Nishiyama et al (1994) Int Arch Allergy Immunol, 105:62-69 Truncated extracellular domain: (aa.23-172) VTLT 50 CNGNNFFEVS STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE 100 SEPVYLEVFS DWLLLQASAE VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE 150 ALKYWYENHN ISITNATVED SGTYYCTGKV WQLDYESEPL NITVIKA Number of amino acids: 151 Molecular weight: 17501.3 Theoretical pI: 4.64 |