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 | Q9GVB7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 605 |
| Molecular Weight | 69896.9 |
| Pi | 5.42 |
| Molecular Weight | 69896.9 |
| Disulphides | 8 |
| Full Sequence |
LLSAFEFTYMINFGRGQNYWEHPYQKSDVYHPINEHREHPKEYEYPLHQEHTYQQEDSGEDENTLQHAYPIDHEGAEPAPQEQNLFSSIEIVERSNYMGNPWTEYMAKYDIEEVHGSGIRVDLGEDAEVAGTQYRLPSGKCPVFGKGIIIENSNTTFLKPVATGNQDLKDGGFAFPPTNPLISPMTLNGMRDFYKNNEYVKNLDELTLCSRHAGNMNPDNDKNSNYKYPAVYDYNDKKCHILYIAAQENNGPRYCNKDQSKRNSMFCFRPAKDKLFENYTYLSKNVVDNWEEVCPRKNLENAKFGLWVDGNCEDIPHVNEFSANDLFECNKLVFELSASDQPKQYEQHLTDYEKIKEGFKNKNASMIKSAFLPTGAFKADRYKSHGKGYNWGNYNRETQKCEIFNVKPTCLINNSSYIATTALSHPIEVEHNFPCSLYKDEIKKEIERESKRIKLNDNDDEGNKKIIAPRIFISDDKDSLKCPCDPEMVSNSTCRFFVCKCVERRAEVTSNNEVVVKEEYKDEYADIPEHKPTYDNMKIIIASSAAVAVLATILMVYLYKRKGNAEKYDKMDQPQHYGKSTSRNDEMLDPEASFWGEEKRASHTT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Giersing B, Miura K, Shimp R, Wang J, Zhou H, Orcutt A, Stowers A, Saul A, Miller LH, Long C, Singh S. (2005) Infection and Immunity, 73, 3963-70 |
| Project Aim | Vaccine studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET24d |
| Expression Protocol | Briefly, fermentation was performed at a 5-liter scale at 37°C using defined medium (KH2PO4 [13.3 g/liter], NH4HPO4 [4.0 g/liter], citric acid monohydrate [1.7 g/liter], MgSO4 · 7H2O [1.2 g/liter], thiamine HCl [4.5 mg/liter], dextrose [25 g/liter], kanamycin [35 mg/liter], and PTM4 trace salts [1 ml/liter]). NH4OH was used to maintain the pH and provide a nitrogen source while glucose was the primary carbon source. At an optical density at 550 nm (OD550) of 35.0, the culture was induced by the addition of isopropyl-1-thio-β-galactopyranoside (IPTG) to a final concentration of 1 mM. Induction continued for 3 hours before harvesting by centrifugation and cell pellet storage at −80°C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 35 = 550 |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 10 mM β-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, 8 M guanidine-HCl, 100 mM NaCl |
| Refolding Buffer | 100 mM Tris-HCl, pH 10.5, 1 mM EDTA, 250 mM NaCl, 200 mM arginine, 1 M urea, 60 mM sucrose, 10 mM cystamine, 4 mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 10.5 |
| Refolding Temperature | 24.0 |
| Protein Concentration | n/a |
| Refolding Time | 12 h |
| Redox Agent | DTT/cystamine |
| Redox Agent Concentration | 4/10 mM |
| Refolding Protocol | The inclusion body pellet was resuspended in solubilization buffer (10 mM β-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, 8 M guanidine-HCl, 100 mM NaCl) and stirred on a magnetic stirrer for 2 h at 4°C. The guanidine-solubilized material was clarified by centrifugation at 25,000 × g for 30 min at 4°C and filtered through a 0.45-μm filter. The denatured supernatant was then refolded by 30-fold rapid dilution in a redox system consisting of refolding buffer (100 mM Tris-HCl, pH 10.5, 1 mM EDTA, 250 mM NaCl, 200 mM arginine, 1 M urea, 60 mM sucrose, 10 mM cystamine, 4 mM DTT). The refolding solution was kept for 24 h at 12°C with continuous stirring and then dialyzed for 48 h against 50 mM Tris-HCl, pH 8.0. The dialyzed solution was clarified by centrifugation and loaded onto an Ni-NTA Superflow (QIAGEN, Valencia, CA) column preequilibrated in 2× phosphate-buffered saline (PBS). The Ni-NTA column was washed with five column volumes of equilibration buffer, and protein was eluted from the column using 1× PBS containing 250 mM imidazole. Fractions containing EcAMA1 were pooled, diluted with 50 mM Tris-HCl, pH 8.0, and applied to a Q-Sepharose Hi Trap column (Amersham Biosciences, Piscataway, NJ) equilibrated with binding buffer (50 mM Tris-HCl, pH 8.0). After sample application, the column was washed with 10 column volumes of binding buffer, and the EcAMA1 was eluted with a linear gradient to 100% of elution buffer (50 mM Tris-HCl, pH 8.0, 1.2 M NaCl). Final purification of refolded EcAMA1 eluted from the Q-Sepharose Hi Trap column was carried out using a Superdex 75 column (Amersham Biosciences) with PBS. |
| Refolding Assay | ELISA,Statistical Analysis |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 200 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |