Refolding Record:
| Protein | |
|---|---|
| Protein Name | Decay accelerating factor (CD55) |
| Abbreviated Name | DAF |
| SCOP Family | Complement control module/SCR domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08174 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 382 |
| Molecular Weight | 41400.1 |
| Pi | 7.78 |
| Molecular Weight | 41400.1 |
| Disulphides | Unknown |
| Full Sequence |
MTVARPSVPA ALPLLGELPR LLLLVLLCLP AVWGDCGLPP DVPNAQPALE GRTSFPEDTV ITYKCEESFV KIPGEKDSVI CLKGSQWSDI EEFCNRSCEV PTRLNSASLK QPYITQNYFP VGTVVEYECR PGYRREPSLS PKLTCLQNLK WSTAVEFCKK KSCPNPGEIR NGQIDVPGGI LFGATISFSC NTGYKLFGST
SSFCLISGSS VQWSDPLPEC REIYCPAPPQ IDNGIIQGER DHYGYRQSVT YACNKGFTMI GEHSIYCTVN NDEGEWSGPP PECRGKSLTS KVPPTVQKPT TVNVPTTEVS PTSQKTTTKT TTPNAQATRS TPVSRTTKHF HETTPNKGSG TTSGTTRLLS GHTCFTLTGL LGTLVTMGLL T
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | White J, Lukacik P, Esser D, Steward M, Giddings N, Bright JR, Fritchley SJ, Morgan BP, Lea SM, Smith GP, Smith RAG (2004) Protein Science, 13, 2406-2415 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | UT5600(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET26b(+) |
| Expression Protocol | 1L of EZMix(Sigma)-kanamycin was inoculated with 1ml of overnight culture in LB medium. Cells were grown for 75min at 37degC (OD600=0.6) and protein was then induced with 1mM IPTG. The culture was grown for a further 3h, and the cells were then harvested by centrifugation (30min x 9000g). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Acid precipitation |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | PBS, 0.05%(w/v) Tween20 |
| Solubilization Buffer | 8M urea, 25mM DTT, 1mM EDTA, 0.1M Tris pH 8.0 |
| Refolding Buffer | 0.02M ethanolamine, 1mM EDTA, 0.5M arginine, 1mM cysteine, 2mM cystine, pH 11.0 |
| Pre-Refolding Purification | Acid precipitation |
| Tag Cleaved | yes |
| Refolding pH | 11.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 32h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | 1mM/2mM |
| Refolding Protocol | Cell pellets were resuspended in 40ml of 20mM TrisHCl, 1mM EDTA pH 8.0 and passed twice through a homogenizer. The homogenate was centrifuged (30min x 10000g, 4degC) and the pellet was washed twice with wash buffer and centrifuged. The pellet was then resuspended in 10ml solubilization buffer and incubated for 2h at room temperature with gentle stirring. The pH was then adjusted to 3-4 with HCl and the solution was centrifuged (30min x 10000g, 4degC). The supernatant was then dialysed in 6M urea, 10mM HCl (pH 3-4) to precipitate the major contaminant (OmpA), which was removed by centrifugation (30min x 10000g, 4degC). The protein was then refolded by rapid dilution (1:50 v/v) with three additions of the same volume of unfolded protein into refolding buffer over a 32h period. Additions were made drop-wise with stirring and the solution was then left static at 4degC. The refolded protein was ultrafiltered and centrifuged (20min x 20000g, 4degC). The protein was then buffer-exchanged into PBS using a G25 Sephadex column. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5mM |
| Refolding Yield | |
| Purity | 80% |
| Notes | A further purification step for crystallization, Superdex S75 run at 18degC, produced >95% pure protein |