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 | O00784 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 628 |
| Molecular Weight | 72622.2 |
| Pi | 5.71362 |
| Molecular Weight | 72622.2 |
| Disulphides | 8 |
| Full Sequence |
HHHHHH MRKLYCVLLL SAFEFTYMIN FGRGQNYWEH PYQNSNVYHP INEHREHPKK YEYPLHQEHT YQQEDSGEDE NTLQHAYPID HEGAEPAPQE QNLFSSIEIV ERSNYMGNPW TEYMAKYDIE EVHGSGIRVD LGEDAEVAGT QYRLPSGKCP
VFGKGIIIEN SNTTFLTPVA TGNQDLKDGG FAFPPTNPPM SPMTLNGMRD LYKNNEYVKN LDELTLCSRH AGNMNPDNDE NSNYKYPAVY DDKDKKCHIL YIAAQENNGP RYCNKDESKR NSMFCFRPAK DKSFQNYTYL SKNVVDNWEE VCPRKNLENA KFGLWVDGNC EDIPHVNEFS ANDLFECNKL VFELSASDQP
KQYEQHLTDY EKIKEGFKNK NASMIKSAFL PTGAFKADRY KSHGRGYNWG NYNRKTQKCE IFNVKPTCLI NNSSYIATTA LSHPIEVEHN FPCSLYKDEI KKEIERESKR IKLNDNDDEG NKKIIAPRIF ISDDIDSLKC PCAPEIVSNS TCNFFVCKCV EKRAEVTSNN EVVVKEEYKD EYADIPEHKP TYDKMKIIIA
SSAAVAVLAT ILMVYLYKRK GNAEKYDKMD EPQHYGKSNS RNDEMLDPEA SFWGEEKRAS HTTPVLMEKP YY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hodder AN, Crewther PE, Anders RF (2001) Int Arch Allergy Immunol, 69, 3286-3294 |
| 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 | BL21 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pDS56/RBS11/6xHis |
| Expression Protocol | details not provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl pH 8.0 |
| Refolding Buffer | 100mM Tris pH 8.0, 1mM reduced glutathione, 0.25mM oxidized glutathione |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.25mM,1mM/0.25mM |
| Refolding Protocol | Protein was produced in E.coli cells and the inclusion bodies isolated (details not provided). The inclusion body pellet was dissolved in solubilization buffer and following centrifugation the supernatant was incubated with Ni-NTA resin for 2h. The unbound fraction contained about 50% of the expressed protein, and therefore was incubated further with second batch of Ni-NTA resin. Following a series of 1- column volume washes with 6M GdnHCl (pH 8.0, 6.3, 5.9), the bound protein was eluted with 6M GdnHCl pH 4.5. The protein was then refolded by dilution into refolding buffer and purified further by anion-exchange chromatography followed by RP HPLC or gel filtration chromatography. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |