| Mossakowska D, Dodd I, Pindar W, Smith RAG
(1999)
Eur J Immunol,
29,
1955-1965 |
| Structure-Function |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3h |
| pDB1013 |
| 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). |
| IPTG |
| OD 600 =
0.5 |
| None |
| None |
| Ion-exchange chromatography |
| insoluble |
| Dilution |
| n/a |
| 20mM Tris, 8M urea, 50mM 2-mercaptoethanol, 1mM EDTA, 0.1mM PMSF pH 8.5 |
| 20mM Tris, 1M urea, 3mM 2-mercaptoethanol, pH 9 |
| Ion-exchange chromatography |
| no tag |
| 9.0 |
| 4.0 |
|
| 3 days |
| Beta-mercaptoethanol |
| 3mM |
| 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 solubilized inclusion bodies were then passed down a Q-sepharose column and the unadsorbed protein was retained. The protein was diluted 16-fold into refolding buffer and left for 3 days at 4degC to refold. It was then concentrated by ultrafiltration |
| SDS-PAGE |
| None |
| None |
|
|
| 70% |
| In this study, 5 different constructs, comprising different numbers of Short Consensus Repeats from N-terminal domain were expressed. This report describes the protocol for SCR3 sequence (a.a.122-196). Protocol for the other sequences are described under a different record for complement receptor 1.
See Dodd et al, 1995, Prot Expr Purif., 6:727-736 for details of expression protocol and inclusion body preparation
|