Refolding Record:
| Protein | |
|---|---|
| Protein Name | HIV protease |
| Abbreviated Name | HIV-PR |
| SCOP Family | Retroviral protease (retropepsin) |
| Structure Notes | |
| Organism | Human |
| 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 | 99 |
| Molecular Weight | 10792.8 |
| Pi | 8.83 |
| Molecular Weight | 10792.8 |
| Disulphides | Unknown |
| Full Sequence |
PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dash C, Sastry M, Rao M. (2005) Biochemistry, 44, 3725-3734 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MC1061 |
| Expression Temp | 42.0 |
| Expression Time | 1 h |
| Expression Vector | pPTN |
| Expression Protocol | Purification of HIV-1 PR and Enzymatic Assay. The Escherichia coli MC1061 containing plasmid pPT∆N containing the recombinant HIV-1 PR that was grown in M9 medium supplemented with 0.2% casamino acids and 50 ug/mL ampicillin. After the onset of the log phase of bacterial growth, the temperature was shifted to 42 °C for 60 min and the bacteria were then pelleted. HIV-1 PR was purified as previously reported (33). Briefly, the bacteria were lysed by sonication and centrifuged at 27000g for 30 min. The protein was purified by ammonium sulfate precipitation, dialysis, and gel-filtration chromatography and stored at -80 °C in the presence of 10% glycerol. The HIV-1 PR activity was assayed using the synthetic substrate Lys-Ala- Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-amide. The HIV-1 PR was incubated at 37 °C with the substrate in a reaction mixture containing 100 mM NaCl, 5 mM B-mercaptoethanol, 5 mM ethylenediaminetetraacetic acid (EDTA), and 50 mM sodium acetate buffer at pH 5.6. After 15 min, the reaction was stopped by the addition of an equal volume of 5% trichloroacetate and followed by incubation for 30 min at 28 °C. The cleavage products were analyzed by RP-HPLC and by a decrease in absorbance at 300 nm (33). |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | RP-HPLC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100 mM NaCl, 5 mM 􏰅-mercaptoethanol,5 mM ethylenediaminetetraacetic acid (EDTA), and 50 mMsodium acetate buffer at pH 5.6 |
| Solubilization Buffer | 50 mM sodium phosphate buffer at pH 6.0 |
| Refolding Buffer | 50 mM sodium phosphate buffer at pH 6.0, GdnHCl (1-8 M) |
| Pre-Refolding Purification | RP-HPLC |
| Tag Cleaved | no tag |
| Refolding pH | 6.0 |
| Refolding Temperature | 28.0 |
| Protein Concentration | n/a |
| Refolding Time | 6-8 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | Denaturation and Renaturation of HIV-1 PR. All experiments described below were performed in the presence of 50 mM sodium phosphate buffer at pH 6.0. For unfolding studies, 100 uM of purified HIV-1 PR was incubated with increasing concentrations of GdnHCl (1-8 M) for 2 h at 28 °C. Renaturation was initiated by diluting 10 µL of the sample into 1 mL of buffer and was mixed for 6-8 h at 28 °C. For the chaperone-assisted refolding, R-crystallin was added at different molar concentrations in the reaction mixture containing 1 uM of HIV-1 PR. CaCl2 or MgCl2 was added at 5 mM to the complexed R-crystallin-HIV-1 PR. As a control, MgCl2 was also added at 5 mM to the complexed R-crystallin-HIV-1 PR. Aliquots (100 uL) were withdrawn at various times of refolding and assayed for proteolytic activity of HIV-1 PR. Reactivation of chemically denatured HIV-1 PR was calculated as the percentage activity relative to a control sample of native HIV-1 PR tested under identical conditions. Additional experimental details are provided in the figure captions. As a control experiment, BSA was added to the renaturation buffer at different molar concentrations to test its ability to refold the unfolded HIV-1 PR. |
| Refolding Assay | Fluorescence |
| Refolding Chaperones | alpha-crystallin |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |