Refolding Record:
| Protein | |
|---|---|
| Protein Name | HIV protease |
| Abbreviated Name | HIV-PR |
| SCOP Family | Retroviral protease (retropepsin) |
| Structure Notes | |
| Organism | HIV-1 |
| UniProt Accession | P03366 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 113 |
| Molecular Weight | 12419.5 |
| Pi | 9.44294 |
| Molecular Weight | 12419.5 |
| Disulphides | 0 |
| Full Sequence |
MRGSHHHGSPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQAGCTLNFRSHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Leuthardt A and Roesel JL (1993) FEBS Letters, 326, 275-280 |
| Project Aim | Drug Studies |
| Fusion | N-terminal and C-terminal poly-histidine tags |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 5h |
| Expression Vector | pDS |
| Expression Protocol | Cells were grown at 37degC in 1L 2xTY medium containing 25microg/ml kanamycin and 100microg/ml ampicillin. When OD600 reached 0.7, the culture was divided in 4x250ml and IPTG added to a final concentration of 400microg/ml. The cultures were induced for up to 5h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM TrisHCl, 50mM NaCl, 10mM EDTA, 0.1mM PMSF, 0.5% (v/v) Triton X-100 pH 8.0 |
| Solubilization Buffer | 50mM TrisHCl, 50mM NaCl, 6M GdnHCl pH 8.0 |
| Refolding Buffer | 20mM MES pH 6.0, 0.01% Triton X-100, 5mM EDTA, 29% glycerol, 10mM DTT |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 6.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 10-30microg/ml |
| Refolding Time | 24h |
| Redox Agent | DTT |
| Redox Agent Concentration | 10mM |
| Refolding Protocol | Cells were centrifuged and the pellet (9-10g/L) was resuspended in 5ml/g of 50mM TrisHCl, 50mM NaCl, 10mM EDTA, 0.1mM PMSF, pH 8.0. Lysozyme was added (1mg/ml) and cells were incubated on ice for 30min and then frozen at -70degC. After thawing, NP-40 was added (0.25%(v/v)), followed by 20min incubation on ice. 0.1mg/ml DNaseI and 10mM MgCl2 were added. The suspension was centrifuged (17000g, 15min) and the insoluble fraction was gently resuspended at 5ml/g in wash buffer, incubated with shaker for 30min at room temperature and centrifuged again. This wash step was repeated twice followed by a wash with an equal volume of deionized water. The pellet was then resuspended at 5mg/ml in solubilization buffer. The inclusion bodies were solubilized overnight with gentle shaking. The unfolded protein was then loaded onto a Ni-iminodiacetic acid column equilibrated with solubilization buffer. The column was washed with solubilization buffer, then the protein was eluted with an imidazole gradient from 0-100mM in solubilization buffer. Selected fractions were diluted to a final concentration of 10-30microg/ml, adjusted to 10mM DTT and dialyzed for 24h against 100x volume refolding buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 29% |
| Refolding Yield | 95% |
| Purity | |
| Notes | |