Refolding Record:
Protein | |
---|---|
Protein Name | CD69 Early activation antigen |
Abbreviated Name | CD69 |
SCOP Family | C-type lectin domain |
Structure Notes | |
Organism | Human |
UniProt Accession | Q07108 |
SCOP Unique ID | n/a |
Structure Solved | |
Class | Alpha+Beta |
Molecularity | Monomer |
Construct | |
---|---|
Full Length | n |
Domain | Extracellular NK-cell domain, aa.82-199 |
Chimera | n/a |
Variants | n/a |
Chain Length | 118 |
Molecular Weight | 13786.5 |
Pi | 8.52383 |
Molecular Weight | 13786.5 |
Disulphides | 3 |
Full Sequence |
VSSCSEDWV GYQRKCYFIS TVKRSWTSAQ NACSEHGATL AVIDSEKDMN FLKRYAGREE HWVGLKKEPG HPWKWSNGKE FNNWFNVTGS DKCVFLKNTE VSSMECEKNL YWICNKPYK
|
Notes | n/a |
Expression | |
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Report | Llera AS, Viedma F, Sanchez-Madrid F, Tormo J (2001) J Biol Chem, 276, 7312-7319 |
Project Aim | Crystallography |
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 | 4h |
Expression Vector | pET26b |
Expression Protocol | Cells were grown in LB medium at 37degC until A600 reached 0.7, expression was induced with 0.5mM IPTG. After 4h, cells were harvested and resuspended in TrisHCl pH 8.0, 0.2M NaCl, 5mM EDTA, 5mM DTT. |
Method of Induction | IPTG |
Cell Density at Induction | OD 600 = 0.7 |
Cell Disruption Method | Sonication |
Lytic Agent | Lysozyme |
Pre-Refolding Purification | None |
Solubility | insoluble |
Refolding | |
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Refolding Method | Dilution |
Wash Buffer | 50mM TrisHCl pH 8.0, 0.1M NaCl, 1mM EDTA, 1mM DTT, 0.5% (v/v) Triton X-100 |
Solubilization Buffer | 25mM MES pH 6.5, 8M urea, 10mM EDTA, 1mM DTT |
Refolding Buffer | 0.1M TrisHCl pH 8.5, 400mM L-arginine, 2mM EDTA, 6.3mM cysteamine, 3.7mM Cystamine, 0.1mM PMSF |
Pre-Refolding Purification | None |
Tag Cleaved | no tag |
Refolding pH | 8.5 |
Refolding Temperature | 4.0 |
Protein Concentration | |
Refolding Time | 36h |
Redox Agent | cysteamine/cystamine |
Redox Agent Concentration | 6.3/3.7mM |
Refolding Protocol | Cells were lysed by adding 1mg/ml lysozyme and sonication. The insoluble inclusion bodies were collected and washed three times in wash buffer and once in 50mM TriHCl pH 8.0, 2M NaCl, 1M urea, 1mM EDTA. The protein was then dissolved in solubilization buffer and the insoluble material discarded by centrifugation. The protein was then refolded by slow dilution to 1L of refolding buffer. Repeated pulses were added every 12h. After 36h, the refolding mixture was concentrated and then purified by gel filtration chromatography in 25mM TrisHCl pH 7.5, 100mM NaCl, 0.1mM EDTA on a Superdex 200 column, followed by cation exchange chromatography using a Mono S column. |
Refolding Assay | Crystallography |
Refolding Chaperones | None |
Refolding Additives | None,L-Arginine |
Additives Concentration | 0.4M |
Refolding Yield | |
Purity | |
Notes |