Refolding Record:
| Protein | |
|---|---|
| Protein Name | ETS1 |
| Abbreviated Name | ETS1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P14921 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 374 |
| Molecular Weight | 42949.8 |
| Pi | 5.57957 |
| Molecular Weight | 42949.8 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSH MKAAVDLKPT LTIIKTEKVD LELFPSPDME CADVPLLTPS SKEMMSQALK ATFSGFTKEQ QRLGIPKDPR QWTETHVRDW VMWAVNEFSL KGVDFQKFCM NGAALCALGK DCFLELAPDF VGDILWEHLE ILQKEDVKPY QVNGVNPAYP ESRYTSDYFI SYGIEHAQCV PPSEFSEPSF ITESYQTLHP ISSEELLSLK YENDYPSVIL RDPLQTDTLQ NDYFAIKQEV VTPDNMCMGR TSRGSGPIQL WQFLLELLTD KSCQSFISWT GDGWEFKLSD PDEVARRWGK RKNKPKMNYE KLSRGLRYYY DKNIIHKTAG KRYVYRFVCD LQSLLGYTPE ELHAMLDVKP DADE
|
| Notes | p42 splice isoform - missing aa.244-330 compared to p51 (full length) isoform |
| Expression | |
|---|---|
| Report | Fisher RJ, Fivash M, Casas-Finet J, Erickson JW, Kondoh A, Bladen SV, Fisher C, Watson DK, Papas T (1994) Protein Science, 3, 257-266 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET15b |
| Expression Protocol | Cells were grown until OD660 reached 1, expression was induced with 1mM ITPG and cells were grown for a further 3h before harvesting. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 660 = 1 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | sequential series of salts, not specified |
| Solubilization Buffer | 8M GdnHCl, 40mM TrisHCl pH 8.5, 100mM 2-mercaptoethanol |
| Refolding Buffer | 10mM Hepes pH 7.3, 3mM EDTA, 0.05% Tween 20, 150mM NaCl |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.3 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 1-10microM |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | Cells were harvested and sonicated, the cell pellet was washed with a sequential series of salts, solubilized in 6M GdnHCl, and purified on a nickel sulfate chelating column. Selected fractions were reduced with 100mM 2-mercaptoethanol, acidified to pH 2.0 with 20% TFA, and purified by further HPLC. The protein was then lyophilized and dissolved in 5ml of denaturation buffer. The protein was then refolded by passage through a Superdex 75 column. |
| Refolding Assay | DNA binding,Surface plasmon resonance binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 71% |
| Purity | n/a |
| Notes | See also Werner et al. 1994, FEBS v.345, p.125-130 |