Refolding Record:
| Protein | |
|---|---|
| Protein Name | Integrase |
| Abbreviated Name | IN |
| SCOP Family | Retroviral protease (retropepsin) |
| Structure Notes | |
| Organism | Equine infectious anemia virus (isolate CL22) (EIA |
| UniProt Accession | P32542 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 233 |
| Molecular Weight | 25755.6 |
| Pi | 8.95 |
| Molecular Weight | 25755.6 |
| Disulphides | Unknown |
| Full Sequence |
CPHCTKQGSGPAGCVMRSPNHWQADCTHLDNKIILTFVESNSGYIHATLLSKENALCTSLAILEWARLFSPKSLHTDNGTNFVAEPVVNLLKFLKIAHTTGIPYHPESQGIVERANRTLKEKIQSHRDNTQTLEAALQLALITCNKGRESMGGQTPWEVFITNQAQVIHEKLLLQQAQSSKKFCFYKIPGEHDWKGPTRVLWKGDGAVVVNDEGKGIIAVPLTRTKLLIKPN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Engelman A (1996) Protein Expression and Purification, 8, 299-304 |
| Project Aim | Identification and Characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET15b |
| Expression Protocol | EIAV IN was expressed in E. coli strain BL21(DE3) (16) as previously described (17). One liter of induced cells was pelleted, resuspended in 40 ml of 25 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (Hepes), pH 7.6 – 1 mM EDTA, and frozen in liquid N2 . The cells were thawed on ice overnight, pelleted, and resuspended in 40 ml of TEM buffer [20 mM Tris – HCl, pH 8.0, 0.1 mM EDTA, 2 mM b-mercaptoethanol (b- ME)] containing 0.5 M NaCl – 5 mM imidazole – 0.2 mg/ ml lysozyme. After 30 min on ice, the lysate was sonicated as described (17, 18) and centrifuged at 40,000g for 30 min, the supernatant (fraction I) was saved, and the pellet was homogenized with 40 ml of TEM buffer containing 2 M NaCl – 5 mM imidazole. The homogenate was stirred at 4C for 30 min and centrifuged at 40,000g for 30 min, the supernatant (fraction II) was saved, and the pellet was homogenized in 20 ml of TEM buffer containing 6 M guanidine – HCl (GndHCl) – 0.5 M NaCl – 5 mM imidazole. The homogenate was stirred at 4C for 30 min and centrifuged at 40,000g for 30 min, and the supernatant (fraction III) was saved. IN-containing fractions were identified following sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS – PAGE) and staining with Coomassie blue. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Tris – HCl, pH 8.0, 0.1 mM EDTA, 2 mM B-mercaptoethanol (B- ME), 6 M GndHCl – 0.5 M NaCl, 25 mM imidazole |
| Solubilization Buffer | 20 mM Tris – HCl, pH 8.0, 0.1 mM EDTA, 2 mM B-mercaptoethanol (B- ME), 6 M GndHCl – 0.5 M NaCl |
| Refolding Buffer | 6 M GndHCl, 20 mM Hepes, pH 7.6, 2 mM EDTA, 2 mM B-ME, and then against 6 M GndHCl,20 mM Hepes, pH 7.6, 2 mM EDTA, 10 mM DTT |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 7.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT/Beta mercaptoethanol |
| Redox Agent Concentration | n/a,10/2 mM |
| Refolding Protocol | For the denatured protein in fraction III, a 1-ml chelating Sepharose FF column was charged and equilibrated with TEM buffer containing 6 M GndHCl – 0.5M NaCl – 5 mM imidazole. Following loading, the column was washed with 10 ml of TEM buffer containing 6 M GndHCl – 0.5 M NaCl – 25 mM imidazole. The column was developed using a 12-ml linear gradient of 25 mM to 0.8 M imidazole in TEM buffer containing 6 M GndHCl – 0.5 M NaCl. EDTA was added to 2 mM to pooled fractions, and the sample was dialyzed first against 6 M GndHCl, 20 mM Hepes, pH 7.6, 2 mM EDTA, 2 mM b-ME, and then against 6 M GndHCl, 20 mM Hepes, pH 7.6, 2 mM EDTA, 10 mM DTT. The concentration of IN was adjusted to 0.5 mg/ml in 3 M GndHCl, 0.5 M NaCl, 20 mM Hepes, pH 7.6, 2 mM EDTA, 10 mM DTT, and the denaturant was removed by stepwise dialysis essentially as described (17, 19). The first dialysis buffer was 2 M urea, 0.5 M NaCl, 20 mM Hepes, pH 7.6, 1 mM EDTA, 1 mM DTT, 10 mM CHAPS; the second was NC buffer; and the third was NC buffer containing 0.5 M NaCl (0.5NC buffer). Soluble IN was recovered at 0.5 mg/ml following centrifuga- tion at 18,500g for 10 min. The protein was treated with thrombin (17), and the thrombin was removed by passage over 0.2 ml benzamidine Sepharose 6B (Pharmacia) equilibrated with 0.5NC buffer. The protein was concentrated by ultrafiltration using an Amicon Centriprep 10 concentrator under conditions specified by the manufacturer. Concentrated IN was dialyzed against 0.5NC buffer and spun at 18,500g for 10 min. Soluble IN (1.0 mg/ml) was liquoted, frozen in liquid N2 , and stored at 080􏰄C. |
| Refolding Assay | Unspecified,enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | |