Refolding Record:
| Protein | |
|---|---|
| Protein Name | HIV-1 Reverse transcriptase |
| Abbreviated Name | HIV-1 RT |
| SCOP Family | Recombinase DNA-binding domain |
| Structure Notes | |
| Organism | HIV |
| UniProt Accession | Q6YA62 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 560 |
| Molecular Weight | 64416.0 |
| Pi | 8.19997 |
| Molecular Weight | 64416.0 |
| Disulphides | 0 |
| Full Sequence |
PISPI ETVPVKLKPG MDGPKVKQWP LTEEKIKALV EICTEMEKEG KISKIGPENP YNTPVFAIKK KDSTKWRKLV DFRELNKRTQ DFWEVQLGIP HPAGLKKKKS VTVLDVGDAY FSVPLDKDFR KYTAFTIPSI NNETPGIRYQ YNVLPQGWKG SPAIFQCSMT KILEPFRKQN PDIVIYQYMD DLYVGSDLEI
GQHRTKIEEL RAHLLSWGFT TPDKKHQKEP PFLWMGYELH PDKWTVQPIM LPEKDSWTVN DIQKLVGKLN WASQIYAGIK VRELCKLLRG TKALTEVVPL TEEAELELAE NREILREPVH GVYYDPSKDL IAEIQKQGLG QWTYQIYQEP YKNLKTGKYA RMRGAHTNDV KQLTEAVQKI STESIVIWGK TPKFKLPIQK
ETWEAWWTEY WQATWIPEWE FVNTPPLVKL WYQLEKDPIV GAETFYVDGA ASRETKLGKA GYVTDRGRQK VVSLTETTNQ KTELHAIHLA LQDSESEVNI VTDSQYALGI IQAQPDRSES EVVNQIIEEL IKKEKVYLSW VPAHKGIGGN EQVDKLVSSG IRKVL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Koller G, Graumann K, Kramer W, Sara M, Jungbauer A (1995) J Chromatography B, 664, 107-118 |
| Project Aim | Undefined |
| 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 | 4-5h |
| Expression Vector | pKKRT66 |
| Expression Protocol | E.coli cells were grown at 37degC in a 20L fermenter with a 14L net volume in M9ZB medium (Per L: 10g bactotryptone, 5g yeast extract, 5g NaCl, 1g NH4Cl, 3g KH2PO5, 6g Na2HPO4, 4g glucose, 0.25g MgSO4). When OD600 reached 1.0 cells were induced with 1mM IPTG at 37deg for a further 4-5 hours and then harvested by centrifugation |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1.0 |
| Cell Disruption Method | Osmotic shock |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl, 100mM TrisHCl pH 8.0, 100mM beta-mercaptoethanol, 1mM EDTA, 20mM NaCl |
| Refolding Buffer | 70mM Tris HCl pH 8.0, 20mM NaCl, 1mM EDTA, 1mM GdnHCl, 2mM reduced glutathione, 0.2mM oxidised glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 88h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/0.2mM,2mM/0.2mM |
| Refolding Protocol | 5g of cell pellet was resuspended in 45ml lysis buffer (5mM EDTA, 20mM TrisHCl pH 8.0, 10mM MgCl2, 10mM monothioglycerol (MTG), 0.5ml/L beta-mercaptoethanol, 0.1% PMSF) containing 1mg/ml lysozyme, 0.015mg/ml RNAseA and 15U/ml DNAseI. After incubation for 10min at 37degC, TritonX-100 was added to a final concentration of 1%. The mixture was then incubated for a further 10min at 37degC and treated with NaCl to a final concentration of 0.5M. The mixture was inucbated again for 10min at 37degC and centrifuged (40000g x 30min, 4degC). The pellet was suspended in 45ml solubilization buffer and stirred overnight at room temperature. The mixture was centrifuged (40000g x 30min, 4degC), and the supernatant was added dropwise to refolding buffer. The ration of protein solution to refolding buffer was 1:25. The refolding solution was stirred gently for 88h at 4degC. The refolding solution was then concentrated using a phenyl sepharose resin equilibrated with Buffer B (10mM sodium phosphate pH 7.3, 1mM EDTA, 1mM MTG, 2% glycerol) containing 2M (NH4)2SO4. The sample was applied repetitively to the column as GdnHCl reduces binding of the protein to the column. The volume of protein loaded onto the column was 0.25x column volume. The column was washed with Buffer B containing 0.8M (NH4)2SO4, and the protein was eluted with Buffer B. The fractions containing the reverse transcriptase were then applied to an AF-heparin Toyopearl 650M column equilibrated with Buffer B containing 150mM NaCl. After washing, the protein was eluted with buffer B containing 250mM NaCl. The protein was then desulted using a Sephadex G50 Coarse column, before being applied to a Fractogel EMD TMAE 650 (S) column equilibrated with Buffer C (50mM glycine, 5% glycerol, pH 9.0). The column was washed with Buffer C, then the protein was eluted stepwise with buffer C containing 50mM, 100mM, 200mM and 1M NaCl |
| Refolding Assay | Western Blot,HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | Protocol for purification of soluble fraction also described (same from Toyopearl column step onwards) - purification of soluble fraction more efficient, better yield of active protein |