Refolding Record:
| Protein | |
|---|---|
| Protein Name | Merozoite surface protein 8 |
| Abbreviated Name | MSP-8 |
| SCOP Family | Merozoite surface protein 1 (MSP-1) |
| Structure Notes | |
| Organism | Plasmodium yoelii |
| UniProt Accession | Q9GSA3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 386 |
| Molecular Weight | 43991.3 |
| Pi | 5.17902 |
| Molecular Weight | 43991.3 |
| Disulphides | Unknown |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSH
MGNCNENGNG NINKANNNSI IRKERKRKSK SDFSKGEPEN KEHEIINLYD DVQELLGPDE MNMLDKYSIL GIDDCSNENE NNKIISEYDL KAMKSVLLYK NRISRASINN LDDVKTVFKR CFNKDDPELS KSYEQIQNQV ANEGTTIIDY LSNYISNIYI KINDEFVKNE EFQLSKYIPE LEIINYVLYN GPKEIGNKIK NELIEINNLI ISESLTSIYS SVVSGLNINC KIKDDLITIL NLANGKYFKV NFSSQATMII PEQYSHESEH MKKISEYFIE
KNRVCKNENC PINSNCYVID SVETCRCIPG FSKNEESENL ECLINESTSC ENNNGGCDVN ANCILLEDKI MCECNNKFNG DGIYCS
|
| Notes | Full sequence of mature protein except for C-terminal anchor sequence |
| Expression | |
|---|---|
| Report | Burns JM Jr, Belk CC, Dunn PD (2000) Infection and Immunity, 68, 6189-6195 |
| Project Aim | Functional 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 | pET15b |
| Expression Protocol | Expression details not provided |
| 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 | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl |
| Refolding Buffer | 50mM TrisHCl pH 8.3, 100mM NaCl, 3mM GSH, 0.3mM GSSG |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3mM/0.3mM,3mM/0.3mM |
| Refolding Protocol | Bacteria pelleted from 250ml culture were resuspended in 15ml 50mM TrisHCl pH 8.0, 100mM NaCl, 10mM EDTA, 10mM DTT, treated with lysozyme and sonicated. The lysate was centrifuged (20min, 25000g), then a 25 to 60% ammonium sulfate fraction of the of the initial lysate was dialysed into 5mM sodium acetate buffer (pH 3.5). The protein was then precipitated a seconde time with ammonium sulfate and then purified by Ni-NTA chromatography in the presence of 6M GdnHCl. The protein was then dialysed into refolding buffer containing 4M GdnHCl. The concentration of GdnHCl in refolding buffer was then sequentially reduced (4, 2, 1, 0.5M) by further dialysis. The final dialysis buffer contained 25mM TrisHCl pH 8.0, 100mM NaCl. |
| Refolding Assay | Bioactivity,Western Blot,Immunoprecipitation |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 1mg/L culture |
| Purity | n/a |
| Notes | n/a |