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 | Ectodomain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 652 |
| Molecular Weight | 73876.1 |
| Pi | 6.9 |
| Molecular Weight | 73876.1 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMDKIISENGDVKFDLIPKENTERSHKLINPWEKFMEKYDIEKVHGSGIRVDLGEDARVENQDYRIPSGKCPVMGKGITIQNSKVSFLTRVATGNQKVREGGLAFPQTDVNISPITIDNLKLMYKDHKEILALNDMSLCAKHASFYVPGTNVNTAYRHPAVYDKSNKTCYILYVAAQENMGPRYCSNEEDNENQPFCFTPEKKDEYKNLSYLTKNLREDWETSCPNKSIQNAKFGVWVDGYCSEYQKKEVHDNKTLLECNQIVFNESASDQPKQYEKHLEDTAKIRRGIVDRNGKLIGEALLPIGSYRADQVKSKGKGYNWANYDKKTKKCYIFNKKPTCLINDKDFVATTALSSLEEGPQESFPCDIYKKKIAEEIKVMNVNRNNNGNDTIKFPRIFISDDKESLNCPCEPTQLTQSTCKFFVCNCVEKRQFISENNEVEIKDEFKSEYESPINQ
LENYKIEKAGFDILMANLETYIKIDEKLEDFVESAEKNKHIASIALNNLNKSGLVTEGES
KKILAKMLNMDAMDLLGIGSNHVCISTNTPENAGCFRYDNGTEEWRCLLGFKKNEDNTGC
VKDDAPVCNNSNNGGCDPNADCREVENTDTTNSKKIVCTCKEPNPNAYYDGVFCSSS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Burns, J. M., Flaherty, R. R., Romero, M. M., Weidanz, W. P. (2003) Vaccine, 21, 1843-1852 |
| Project Aim | Vaccine 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 | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pET-15b |
| Expression Protocol | Induced cells grown in 250ml culture were harvested and resuspended in 15ml of TNE buffer (50mM TrisHCl, pH 8.0, 100mM NaCl, 10mM EDTA) and lysed by sonication follow treatment with lysozyme. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | A. 50mM TrisHCl, pH 8.0, 100mM NaCl, 10mM EDTA, 1% deoxycholate. B. 2M urea, 50mM TrisHCl, pH 8.0, 100mM NaCl, 10mM EDTA |
| Solubilization Buffer | 100mM TrisHcl, pH 8.5, 10mM EDYA, 25mM DTT, 8M urea |
| Refolding Buffer | 50mM Tris-HCl, pH 8.3, 100mM NaCl, 3mM reduced glutathione, 0.3mM oxidized glutathione |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.3 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were isolated by centrifugation(2000g, 10min) and washed twice with washing buffer A, followed by a further two times with washing buffer B. The pellet was solubilized and then dialyzed and purified by ammonium sulfate fractioning. The protein then underwent nickel chelate chromatography under denaturing conditions in 6M guanidine HCl, and was refolded by the gradual removal of guanidine HCl by dialysis into refolding buffer. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 8-10mg/l |
| Purity | n/a |
| Notes | n/a |