Refolding Record:
| Protein | |
|---|---|
| Protein Name | Complement receptor type1 |
| Abbreviated Name | CR1 |
| SCOP Family | Complement control module/SCR domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P17927 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Short consensus repeat modules 1-3, aa1-196 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 196 |
| Molecular Weight | 21696.6 |
| Pi | 8.26534 |
| Molecular Weight | 21696.6 |
| Disulphides | >10 |
| Full Sequence |
QCNAPEWLP FARPTNLTDE FEFPIGTYLN YECRPGYSGR PFSIICLKNS VWTGAKDRCR RKSCRNPPDP VNGMVHVIKG IQFGSQIKYS CTKGYRLIGS SSATCIISGD TVIWDNETPI CDRIPCGLPP TITNGDFIST NRENFHYGSV VTYRCNPGSG
GRKVFELVGE PSIYCTSNDD QVGIWSGPAP QCIIPNK
|
| Notes | |
| Expression | |
|---|---|
| Report | Dodd I, Mossakowska DE, Camilleri P, Haran M, Hensley P, Lawlor EJ, McBay DL, Pindar W, Smith RAG (1995) Protein Expression and Purification, 6, 727-736 |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pDB1013 |
| Expression Protocol | Cells were grown in 500ml NCYZM medium (1%(w/v) bactotryptone, 0.5%(w/v) bactoyeast, 0.5%(w/v) NaCl, 0.1%(w/v) casamino acids, 0.2%(w/v) MgSO4.7H2O, pH 7.2) containing 150microg/ml ampicillin. Cells were grown until A600 reached 0.5, expression was induced with 1mM IPTG and cells were grown for a further 3h. the cell were harvested by centrifugation (8000g, 10min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris, 8M urea, 50mM 2-mercaptoethanol, 1mM EDTA, 0.1mM PMSF pH 8.5 |
| Refolding Buffer | 0.02M ethanolamine/1mM EDTA |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 3 days |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.5mM |
| Refolding Protocol | The frozen cell pellet was allowed to thaw at 4deg for 2h and resuspended in 50mM Tris, 50mM NaCl, 1mM EDTA, 0.1mM PMSF pH 8.0. The suspension was sonicated and then centrifuged (6000g, 4degC, 10min). The pellet was the resuspended in solubilization buffer and left at room temperature for 1-1.5h. The protein was added to SP-sepharose, the mixture was swirled and then left at room temperature for 1-2h. The supernatant was removed, then the slurry was poured into a column. The column was equilibrated and washed with 0.02M Tris, 8M urea, 0.05M 2-mercaptoethanol, 0.001M EDTA pH 8.5 at 4deg. The protein was then eluted with the same buffer containing 1M NaCl, The protein was then diluted with 0.02M, 8M urea, 1M NaCl, 0.05M 2-mercaptoethanol pH 8.5 to 2mg/ml. 30 mL of the protein was then added over 1min to 930ml freshly prepared refolding buffer and was left for 1h at 4degC. 1mM reduced glutathione and 0.5mM oxidased glutathione were added, then the solution was left for 3days at approximately 2-3degC. The solution was then ultrafiltrated and further purified using a Toyopear Butyl column. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | Also in Mossakowska et al, 1999, Eur J Immunol, 29:1955-1965, other constructs also expressed and refolded in a similar manner SCR(1-2) a.a.1-124 SCR(1-4) a.a.1-253 SCR(3-4) a.a.122-263 |