Refolding Record:
| Protein | |
|---|---|
| Protein Name | Erythrocyte membrane protein 1 |
| Abbreviated Name | var2CSA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | Q6UDW7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | DBL2-X (residues 530-863) and DBL3-X (residues 1221-1548) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 661 |
| Molecular Weight | 75169.9 |
| Pi | 8.96 |
| Molecular Weight | 75169.9 |
| Disulphides | Unknown |
| Full Sequence |
LEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNN FGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSP WSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSLNATNYIRGCQSKTYDGKIFPGKGGEKQWICKDTIIHGDTNGACIPPRTQNLCVGELWDKSYGGRSNIKNDTKELLKEKIKNAIHKETELLYEYHDTGTAIISKNDKKGQKGKNDPNGLPKGFCHAVQRSFIDYKNMILGTSVNIYEHIGKLQEDIKKIIEKGTPQQKDKIGGVGSSTENVNAWWKGIEREMWDAVRCAITKINKKNNNSIFNGDECGVSPPTGNDEDQSVSWFKEWGEQFCIERLRYEQNIREACTINGKNEKKCINSKSGQGDKIQGACKRKCEKYKKYISEKKQEWDKQKTKYENKYVGKSASDLLKENYPECIS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fernandez P, Viebig NK, Dechavanne S, Lepolard C, Gysin J, Scherf A, Gamain B. (2008) Malar J., 7, Epub ahead of p |
| Project Aim | Vaccine studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | BL21 (DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET24a |
| Expression Protocol | Synthetic genes encoding for var2CSA DBL2-X and DBL3-X were designed with optimized codons for E. coli expression. Synthetic genes encoding FCR3 var2CSA (accession AY372123) DBL2-X (residues 530-863) and DBL3-X (residues 1221-1548) domains fused to a His6-tag on the C-terminus were cloned into the pET24a expression vector between the NdeI and EcoRI restriction sites. Transformed E. coli BL21 (DE3) cells were grown at 37 °C in LB medium with 30 µg/ml of kanamycin to an absorbance of 0.5 at 600 nm, and were then induced with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h at 37 °C under good aeration. Cells were harvested by centrifugation at 6,000 g. The pellets from 800 ml of culture were resuspended in 30 ml of 50 mM Tris.HCl, pH 8.5, containing 150 mM NaCl, in the presence of protease inhibitors. The cells were disrupted by sonication on ice and the suspensions were centrifuged for 20 min at 5,000 g. Pellets were resuspended and washed twice with 30 ml of 50 mM Tris.HCl, pH 8.5, containing 150 mM NaCl, in the presence of protease inhibitors, and finally centrifuged for 20 min at 10,000 g. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris.HCl, pH 8.5, containing 150 mM NaCl |
| Solubilization Buffer | 50 mM Tris.HCl pH 8.5 buffer, containing 150 mM NaCl, 8 M Urea and 5 mM |
| Refolding Buffer | 50 mM Tris.HCl pH 8.5, 20 mM NaCl, 0.4 mM KCl, 0.5 % Triton-X-100 and 0.5 mM DTT |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.5 mM |
| Refolding Protocol | The pellets containing DBL2-X or DBL3-X inclusion bodies were then denatured overnight at 25 °C under agitation in 50 mM Tris.HCl pH 8.5 buffer, containing 150 mM NaCl, 8 M Urea and 5 mM Dithiothreitol (DTT). The suspensions were centrifuged for 30 min at 15,000 g and the pellet was discarded. Refolding was assayed with the AthenaESTM kit (Athena environmental Sciences, Inc.) according to the manufacturer’s instructions. Once the best condition was established, denatured proteins were immobilized on a HisTrap FF Ni-affinity column previously equilibrated with the same buffer, and connected to an FPLC Akta System. The proteins were refolded on the column by adding quickly the refolding buffer (50 mM Tris.HCl pH 8.5, 20 mM NaCl, 0.4 mM KCl, 0.5 % Triton-X-100 and 0.5 mM DTT). After extensive washing with the refolding buffer without Triton-X-100, the protein was eluted with an imidazole gradient (0 to 0.5 M) and aliquots containing purified DBL domains were pooled. After dialysis against 0.9 % NaCl and concentration by means of Macro- and Microsep concentrators, a further stage of gel filtration (Superdex 75, Amersham Pharmacia Biotech) was required to separate the monomeric proteins from the aggregated material and other impurities. Purified DBLs were subsequently concentrated by means of Macro- and Micro-sep concentrators. Protein concentration was determined using the Bio-Rad protein assay. Purity of the samples was checked by SDS-PAGE and Western blot. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Triton X-100 |
| Additives Concentration | 0.5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |