Refolding Record:
| Protein | |
|---|---|
| Protein Name | Uncoupling protein 2 |
| Abbreviated Name | UCP2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P55851 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 309 |
| Molecular Weight | 33229.4 |
| Pi | 9.73678 |
| Molecular Weight | 33229.4 |
| Disulphides | 0 |
| Full Sequence |
MVGFKATDVP PTATVKFLGA GTAACIADLI TFPLDTAKVR LQIQGESQGP VRATASAQYR GVMGTILTMV RTEGPRSLYN GLVAGLQRQM SFASVRIGLY DSVKQFYTKG SEHASIGSRL LAGSTTGALA VAVAQPTDVV KVRFQAQARA GGGRRYQSTV NAYKTIAREE GFRGLWKGTS PNVARNAIVN CAELVTYDLI
KDALLKANLM TDDLPCHFTS AFGAGFCTTV IASPVDVVKT RYMNSALGQY SSAGHCALTM LQKEGPRAFY KGFMPSFLRL GSWNVVMFVT YEQLKRALMA ACTSREAPF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jekabsons MB, Echtay KS, Brand MD (2002) Biochem J., 366, 565-571 |
| 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 | C41 |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pET |
| Expression Protocol | Cells were incubated in TB media with 100 micrograms/ml ampicillin at 37degC at 250rpm until OD600 reahced 0.5-0.6. Protein expression was induced by addition of 1mM IPTG and incubated for a further 2 hours. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.6 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Centrifugal filtration |
| Wash Buffer | n/a |
| Solubilization Buffer | 5mM MOPS, 30mM Na2SO4, 1.5% sarkosyl, pH 7.3 |
| Refolding Buffer | 5mM MOPS, 30mM Na2SO4, pH 7.3 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cell pellets were solubilized in solubilization buffer for 45-60min at 20-22degC. Insoluble material was removed by centrifugation at 27200g for 15min. Refolding was performed by gradual simultaneous removal of sarkosyl and addition of non-ionic detergent (C8E5, C10E6, C12E9, C13E10 or digitonin) by centrifugal filtration. Sequential 70-90 microlitre aliquots of non-ionic detergent (0.1-1% (v/v) in refolding buffer) were mixed with the colubilized inclusion bodies and centrifuged at 12000g for 5-9min at 4degC. This was repeated 15 times, rollowed by a further 12 additions of refolding buffer until the calculated concentration of sarkosyl was 0.01% or less. The final sample was centrifuged at 16000g for 30 min at 4degC to remove the insoluble material. The protein was then loaded in C12E9 onto a hydroxyapatite column which was equilibrated with refolding buffer. Fractions containing protein were pooled and slowly concentrated to approximately 2.5mg/ml using centrifugal filtration. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | >14-18% |
| Purity | |
| Notes | Different detergents used with hydroxyapatite column - best yield with C12E9. |