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 | Q7KQK5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | 3 subdomains of main ectodomain, aa.83-531 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 479 |
| Molecular Weight | 54634.3 |
| Pi | 5.97 |
| Molecular Weight | 54634.3 |
| Disulphides | 8 |
| Full Sequence |
MAHHHHHHPGGSGSGTMH GAEPAPQE QNLFSSIEIV 100
ERSNYMGNPW TEYMAKYDIE EVHGSGIRVD LGEDAEVAGT QYRLPSGKCP VFGKGIIIEN SNTTFLTPVA TGNQYLKDGG FAFPPTEPLM SPMTLDEMRH FYKDNKYVKN LDELTLCSRH AGNMIPDNDK NSNYKYPAVY DDKDKKCHIL YIAAQENNGP RYCNKDESKR NSMFCFRPAK DISFQNYTYL SKNVVDNWEK VCPRKNLQNA KFGLWVDGNC EDIPHVNEFP AIDLFECNKL VFELSASDQP KQYEQHLTDY EKIKEGFKNK NASMIKSAFL PTGAFKADRY KSHGKGYNWG NYNTETQKCE IFNVKPTCLI NNSSYIATTA LSHPIEVENN FPCSLYKDEI MKEIERESKR IKLNDNDDEG NKKIIAPRIF ISDDKDSLKC PCDPEMVSNS TCRFFVCKCV ERRAEVTSNN EVVVKEEYKD E AAALEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dutta S, Lalitha PV, Ware LA, Barbosa A, Moch JK, Vassell MA, Fileta BB Kitov S, Kolodny N, Heppner DG, Haynes JD, Lanar DE (2002) Int Arch Allergy Immunol, 70, 3101-3110 |
| Project Aim | Functional Studies |
| Fusion | N-terminal and C-terminal poly-histidine tags |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | Origami |
| Expression Temp | 27.0 |
| Expression Time | 1hr |
| Expression Vector | modified pET32 |
| Expression Protocol | Medium consisting of Super Broth containing 0.8& hlycerol and 12.5mg/mL of tetracycline was inoculated with an overnight culture. Incubation conditions were maintained at 27degC, pH 7.2 and 400rpm. When OD600 reached 7.0, IPTG was added to a final concentration of 0.1mM and the culture was incubated for a further 1 hour. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 600 = 7 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 15mM Na2HPO4, 5.1mM KH2PO4, 450mM NaCl, pH 7.4, 5% sodium N-lauroylsarcosine |
| Refolding Buffer | 20mM sodium phosphate, 1mM EDTA, 1mM reduced glutathione, 0.25mM oxidized glutathione, pH 8.0 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | |
| Refolding Time | 15h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.5mM |
| Refolding Protocol | Cells were harvested by centrifugation and then resuspended in 5x (wg/vol) buffer A (15mM Na2HPO4, 5.1mM KH2PO4, 450mM NaCl, pH 7.4). Sodium N-lauroylsarcosine (Sarkosyl) was added to a final concentration of 5% (v/v). The suspension was mixed and the cells were disrupted by high-pressure microfluidization. The cell lysate was centrifuged (22000g) and the supernatant was diluted four-fold into Buffer A and loaded onto a Ni-NTA column (0.5ml packed resin per g of paste)pre-equilibrated with Buffer B (buffer A containing 1.25% sarkosyl, pH 7.4). The column was then washed with 20 column volumes (CV) of buffer C (buffer A with 10mM imidazole, 0.125% Sarkosyl pH 7.4) and then 20CV of buffer D (20mM Na phosphate, 25mM imidazole, 0.125% sarkosyl, pH 8.0). Bound proteins were then eluted in Buffer D containing 500mM imidazole (pH 8.0). The eluted protein was diluted 40-fold into refolding buffer. Refolding was performed at room temperature (~22degC) for at least 15h under nitrogen pressure. The refolded protein was then loaded onto a DEAE column pre-equilibrated with refolding buffer +/- GSH/GSSG. The column was then washed with at least 30CV of the buffer followed by 10CV of buffer F (5mM sodium phosphate, 50mM NaCl, 1mM EDTA pH 8.0) The protein was then eluted in buffer F containing 100mM NaCl. The eluted protein was pH adjusted to 5.7 with 1M NaH2PO4, then loaded onto an SP sepharose column equilibrated with buffer G (50mM sodium phosphate, 0.1mM EDTA, 100mM NaCl pH 5.7). The column was washed with 20CV of buffer G containing 275mM NaCl followed by 10CV of a pH exchange buffer (5mM sodium phosphate, 0.1mM EDTA pH 7.1). The protein was then eluted in 23.5mM NaH2PO4.H2O, 37.5mM NaCl, 0.1mM EDTA, pH 7.1). |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | >99% |
| Notes | - Different refolding conditions tested to optimise protocol - eg.reduction of nickel-eluted protein with 5mM DTT for 1h at 37degC prior to refolding (no effect), different GSH/GSSG ratios 1/0.1 and 1/0.25 equally efficient |