Refolding Record:
| Protein | |
|---|---|
| Protein Name | Apical membrane antigen 1 |
| Abbreviated Name | AMA1 |
| SCOP Family | Apical membrane antigen 1 |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | A3F8A6 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | AMA1 domain II |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 127 |
| Molecular Weight | 14471.3 |
| Pi | 7.09 |
| Molecular Weight | 14471.3 |
| Disulphides | Unknown |
| Full Sequence |
AKFGLWVDGNCEDIPHVNEFPAIDLFECNKLVFELSASDQPKQYEQHLTDYEKIKEGFKNKNASMIKSAFLPTGAFKADRYKSHGRGYNWGNYNTKTQKCEIFNVKPTCLINNSSYIATTALSHPIE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Feng ZP, Keizer DW, Stevenson RA, Yao S, Babon JJ, Murphy VJ, Anders RF, Norton RS. (2005) J Mol Biol, 350, 641-656 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 pLysS |
| Expression Temp | 0.0 |
| Expression Time | 3 h |
| Expression Vector | n/a |
| Expression Protocol | A clone of PfAMA1 domain II, provided by Tony Hodder, was transformed into E. coli strain BL21 DE3 PlysS cells (Stratagene, CA). For unlabelled protein, expression was performed in baffled flasks with cells grown to an A600 of 0.6 in Superbroth containing 2% (w/v) glucose and 100 μg ml−1 of ampicillin. Expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were harvested three hours after induction by centrifugation at 6200 g and 4 °C for 30 minutes. For 15N labelling, cells were grown to an A600 of 0.6 in M9 medium containing 1.0 g l−1 of 15NH4Cl as the sole nitrogen source. For 15N/13C-labelled samples 1.0 g l−1 of 15NH4Cl and 4 g l−1 of [13C]glucose were the sole nitrogen and carbon sources, respectively. Cells were harvested eight hours after IPTG induction by centrifugation at 6200 g and 4 °C for 30 minutes. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M GdnHCl (pH 8.0) |
| Refolding Buffer | 1 mM reduced glutathione and 0.25 mM oxidised glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.25 mM |
| Refolding Protocol | The induced E. coli pellet was solubilised in 6 M GdnHCl (pH 8.0), and purified essentially as described.22 The protein was then refolded by dilution in a buffer containing 1 mM reduced glutathione and 0.25 mM oxidised glutathione as described.5 The refolded protein was diluted 1:4 into buffer A (10 mM NaPO4, 50 mM NaCl (pH 6.0)) and purified by cation-exchange chromatography. Bound protein was eluted from a 5 ml Hitrap SPFF cation-exchange column (Amersham Biosciences, Uppsala, Sweden) using a linear gradient from 0% to 100% buffer B (buffer A with 1 M NaCl) over 30 minutes at a flow rate of 3 ml min−1.Purified and refolded protein samples were analysed for mass by matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) mass spectrometry (Voyager Biospectrometry PR system; Applied Biosystems) with a sinapinic acid matrix.Refolding of PfAMA1 DII was monitored by RP-HPLC. A total of 25 μg of pre-refolded starting material (SM), alkylated refolded material (RA) or refolded material (R) was run on RP-HPLC (column: Brownlee, Aquapore RP-300 7 μ; 100 mm×2.1 mm) over a gradient of 0–100% buffer b for 12 minutes and the resulting chromatograms compared (data not shown). Reversed-phase HPLC was performed using a Hewlett–Packard (Waldbronn, Germany) 1050 modular HPLC. Instrument control, data acquisition, and evaluation were performed using Hewlett–Packard HPLC3D Chemstation software. Buffer a comprised 0.05% (v/v) trifluoroacetic acid (HPLC/Spectro grade; Pierce, Rockford, IL) in Milli-Q grade water (Millipore, Bedford, MA); buffer b comprised 0.05% (v/v) trifluoroacetic acid in acetonitrile (ChromAR HPLC grade; Mallinckrodt, Paris, KY) in Milli-Q grade water. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |