Refolding Record:
| Protein | |
|---|---|
| Protein Name | Apical membrane antigen 1 |
| Abbreviated Name | AMA1 |
| SCOP Family | Apical membrane antigen 1 |
| Structure Notes | |
| Organism | Plasmodium chabaudi |
| UniProt Accession | P16445 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 464 |
| Molecular Weight | 53371.1 |
| Pi | 6.72298 |
| Molecular Weight | 53371.1 |
| Disulphides | 8 |
| Full Sequence |
HHHHHH EGTDKIIS ENGDVKFDLI PKENTERSHK LINPWEKFME
KYDIEKVHGS GIRVDLGEDA RVENQDYRIP SGKCPVMGKG ITIQNSKVSF LTRVATGNQK VREGGLAFPQ TDVNISPITI DNLKLMYKDH KEILALNDMS LCAKHASFYV PGTNVNTAYR HPAVYDKSNK TCYILYVAAQ ENMGPRYCSN EEDNENQPFC FTPEKKDEYK NLSYLTKNLR EDWETSCPNK SIQNAKFGVW VDGYCSEYQK KEVHDNKTLL ECNQIVFNES ASDQPKQYEK HLEDTAKIRR GIVDRNGKLI GEALLPIGSY RADQVKSKGK GYNWANYDKK TKKCYIFNKK PTCLINDKDF VATTALSSLE EGPQESFPCD IYKKKIAEEI KVMNVNRNNN GNDTIKFPRI FISDDKESLN CPCEPTQLTQ STCKFFVCNC VEKRQFISEN NEVEIKDEFK SEYESPINQR
|
| Notes | Estimated sequence only, exact boundaries not clear from paper |
| Expression | |
|---|---|
| Report | Anders RF, Crewther PE, Edwards S, Margetts M, Matthew MLSM Pollock B, Pye D (1998) Molecular Immunology, 16, 240-247 |
| Project Aim | Drug 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 | JPA101 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pDS56/RBSii,6xHis |
| Expression Protocol | no details provided |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Triton X-100, EDTA (concentrations not specified) |
| Solubilization Buffer | 6M GdnHCl, 10mM TrisHCl pH 8.0 |
| Refolding Buffer | 100mM TrisHCl pH 8.0, 1 microM reduced glutathione, 0.25 microM oxidized glutathione |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.05mg/ml |
| Refolding Time | 16h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1microM/0.25microM |
| Refolding Protocol | Expression by E.coli cells were induced with IPTG (details not provided) and were harvested by centrifugation. The cells were lysed with lysozyme and sodium deoxycholate in the presence of PMSF and then centrifuged. The insoluble inclusion bodies were washed with Triton X-100 and EDTA (concentrations not specified) and then incubated for >1h in solubilization buffer. The mixture was then centrifuged and filtered and then loaded onto a 10ml Ni-NTA resin column. The column was washed successively with solubilization buffer at pH 8.0, pH 6.3 and pH 5.9, then the protein was eluted in solubilization buffer at pH 4.5. The protein was refolded by dilution in 100 volumes of ice-cold refolding buffer without oxidized glutathione. After the refolding mixture was incubated on ice for at least 2min, oxidized glutathione was added and the mixture was incubated under nitrogen at room temperature for 16h. After dialysis overnight against 10mM TrisHCl pH 8.0, the refolded protein was incubated with 10mM DEAE-sepharose equilibrated with 10mM TrisHCl pH 8.0. After washing, the refolded protein was eluted with 0.5M NaCl, 10mM TrisHCl, pH 8.0 |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |