Refolding Record:
| Protein | |
|---|---|
| Protein Name | HIV protease |
| Abbreviated Name | HIV-PR |
| SCOP Family | Retroviral protease (retropepsin) |
| Structure Notes | |
| Organism | HIV |
| UniProt Accession | P03366 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | tethered dimer: protease domains tethered head-to-tail |
| Variants | n/a |
| Chain Length | 204 |
| Molecular Weight | 21796.8 |
| Pi | 9.13863 |
| Molecular Weight | 21796.8 |
| Disulphides | 0 |
| Full Sequence |
VTLWQRPLV TIKIGGQLKE ALLGTGADDT VLEEMSLPGR
WKPKMIGGIG GFIKVRQYDQ ILIEICGHKA IGTVLVGPTP VNIIGRNLLT QIGCTLNFPI GGSSG VTLWQRPLV TIKIGGQLKE ALLDTGADDT VLEEMSLPGR WKPKMIGGIG GFIKVRQYDQ ILIEICGHKA IGTVLVGPTP VNIIGRNLLT QIGCTLNFPI
|
| Notes | leader sequence auto-cleaved prior to unfolding and refolding |
| Expression | |
|---|---|
| Report | Cheng YE, Yin FH, Foundling S, Blomstrom D, Kettner CA (1990) Proc. Natl. Acad. Sci. USA, 87, 9660-9664 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM105 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pET3 |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 67% acetic acid |
| Refolding Buffer | 20mM Mes, 1mM DTT, 10% glycerol, pH 5.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 5.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 2h |
| Redox Agent | DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | After expression, cells were harvested and lysed using a French pressure cell. Inclusion bodies were extracted with 67% acetic acid, then diluted 33-fold with water. The extracts were then dialyzed against water overnight, then dialyzed for 2h at 25degC against refolding buffer. The refolded protein was then concentrated, loaded onto a Sephadex G-75 column and eluted with 0.1% acetic acid/1.0mM DTT/10% glycerol. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |