Refolding Record:
| Protein | |
|---|---|
| Protein Name | ATP synthase F1 beta subunit |
| Abbreviated Name | ATP synthase F1 beta subu |
| SCOP Family | RecA protein-like (ATPase-domain) |
| Structure Notes | |
| Organism | Spinacia oleracea |
| UniProt Accession | P00825 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 498 |
| Molecular Weight | 53744.6 |
| Pi | 5.22 |
| Molecular Weight | 53744.6 |
| Disulphides | 0 |
| Full Sequence |
MRINPTTSDP GVSTLEKKNL GRIAQIIGPV LDVAFPPGKM PNIYNALIVK GRDTAGQPMN VTCEVQQLLG NNRVRAVAMS ATDGLTRGME VIDTGAPLSV PVGGATLGRI FNVLGEPVDN LGPVDTRTTS PIHRSAPAFT QLDTKLSIFE TGIKVVDLLA PYRRGGKIGL FGGAGVGKTV LIMELINNIA KAHGGVSVFG GVGERTREGN DLYMEMKESG VINEQNIAES KVALVYGQMN EPPGARMRVG LTALTMAEYF RDVNEQDVLL FIDNIFRFVQ AGSEVSALLG RMPSAVGYQP TLSTEMGSLQ ERITSTKEGS ITSIQAVYVP ADDLTDPAPA TTFAHLDATT VLSRGLAAKG IYPAVDPLDS TSTMLQPRIV GEEHYEIAQR VKETLQRYKE LQDIIAILGL DELSEEDRLT VARARKIERF LSQPFFVAEV FTGSPGKYVG LAETIRGFQL ILSGELDSLP EQAFYLVGNI DEATAKAMNL EMESKLKK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chen Z, Wu I, Richter ML, Gegenheimer P. (1992) FEBS Letters, 298, 69-73 |
| 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 | 5h |
| Expression Vector | pET3a |
| Expression Protocol | Cells were grown at 37degC in LB medium containing ampicillin and chloramphenicol. In mid-exponential phase growth, cells were induced with 0.1mM IPTG for up to 5h, then harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl, 2mM EDTA, pH 8.0 |
| Solubilization Buffer | 4M urea, 50mM Tris-HCl, 2mM EDTA, 2mM DTT, pH 8.0 |
| Refolding Buffer | 50mM Tris-HCl, 2mM EDTA, 2mM DTT, 1mM ATP, 5-20% (v/v) glycerol, 3-0M urea |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | 2mM |
| Refolding Protocol | Urea was removed by three sequential steps of dialysis against buffered solutions containing decreasing amounts of urea. The first step was dialysis for 7h against 100 vols of buffer containing 5% glycerol, 3M urea. The second step was dialysis for 10h against 100 vols of buffer containing 10% glycerol, 1.5M urea. The last step was dialysis for 7h at room temp against 125 vols of buffer containing 20% glycerol, 0M urea. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 5-20% |
| Refolding Yield | |
| Purity | pure |
| Notes | |