Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rapamycin target protein |
| Abbreviated Name | TOR |
| SCOP Family | FKBP12-rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P42345 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | FRB domain (2014-2114) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 98 |
| Molecular Weight | 11869.6 |
| Pi | 5.8 |
| Molecular Weight | 11869.6 |
| Disulphides | Unknown |
| Full Sequence |
EELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dames SA. (2008) Protein Expression and Purification, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | Rosetta (DE3) |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pET-11a |
| Expression Protocol | Expression of hFRB hFRB was expressed from Escherichia coli Rosetta (DE3) cells (Novagen). One hundred milliliters of LB medium supplemented with 100 μg/ml ampicillin and 40 μg/ml chloramphenicol were inoculated with a single colony of transformed cells and grown overnight at 30 °C. The starter culture was diluted 1:40 in 2 L LB medium or 1:20 in 2 L M9 minimal medium, both supplemented with 100 μg/ml ampicillin and 40 μg/ml chloramphenicol. For growth in minimal medium the cells were centrifuged gently for 5 min at 2000 rpm. The supernatant was discarded and the cells were resuspended in minimal medium. The prepared culture was grown at 37 °C to an OD600 of ≈0.8 and induced with 1 mM IPTG. The cells continued growing overnight at 37 °C. Uniformly 15N-labeled hFRB was prepared in minimal medium containing 15NH4Cl as the sole nitrogen source. The used M9 minimal medium contained M9 salts w/o NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, 1× BME vitamin solution (Sigma), 4 g/L glucose, and 1 g/L 15NH4Cl.Cells expressing hFRB were harvested by centrifugation at 4 °C (20 min, 5000 rpm, Sorvall SLA-3000 rotor). The cell pellet was resuspended in 50 ml lysis buffer (50 mM Tris, 2 mM EDTA, 2 mM benzamidine, 2 mM DTT, pH 8). The cell suspension was supplemented with 1 ml of lysozyme stock solution (10 mg/ml) and incubated for 10 min at room temperature. Cells were disrupted by sonication on ice using a Branson Digital Sonifier and a power level of 40%. During sonication for 6 min, intervals of 3 s pulsing alternated with 7 s breaks. To verify that hFRB is in the inclusion body fraction 100 μl of the sonicated cell suspension were centrifuged and the supernatant and the pellet checked by SDS–PAGE. After that the whole sonicated cell suspension was centrifuged for 30 min at 12,000 rpm (Sorvall SS34 rotor). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | RP-HPLC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris, 1 M urea, 10 mM TCEP, pH 8 |
| Solubilization Buffer | 50 mM Tris, 6 M GdmCl, 10 mM TCEP |
| Refolding Buffer | 20 mM Tris, 1 mM TCEP, 300 mM NaCl, pH 8 |
| Pre-Refolding Purification | RP-HPLC |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The supernatant was discarded and the pellet was washed twice with 20 mM Tris, 1 M urea, 10 mM TCEP, pH 8. The washed inclusion body pellet was extracted with 20 ml, 50 mM Tris, 6 M GdmCl, 10 mM TCEP, pH 8 by disrupting the pellet manually and incubating it for 1 h at 4 °C. After extraction the mixture was centrifuged for 30 min at 32,000 rpm and 4 °C using an ultracentrifuge. Protein samples containing 6 M GdmCl were precipitated in the following way to check them by SDS–PAGE: Mix 30 μl protein solution with 30 μl H2O and 60 μl 10% TCA and leave on ice for 20 min, centrifuge for 15 min in a microfuge (13,000 rpm), discard the supernatant and wash the pellet with 60 μl ice cold ethanol, dry the pellet from ethanol and resuspend it in 60 μl 2× sample buffer. To further purify hFRB, the inclusion body extract was first diluted 1:3 with inclusion body extraction buffer and then 2:1 with 30% HPLC buffer B (90% acetonitrile/0.1%TFA) and applied to a preparative C4 HPLC column that had been equilibrated with 30% HPLC buffer B. HPLC buffer A was 0.1% TFA in water. To elute the protein a gradient was started that increased the concentration of buffer B by 1.4%/min; hFRB eluted around 52–60% B. Two hundred microliters of each fraction were dried in a SpeedVac and dissolved in 20 μl 2× sample buffer to analyze them by SDS–PAGE. Those containing nearly pure hFRB were frozen in liquid nitrogen, lyophilized and stored at −20 °C. The protein concentration in each fraction was determined based on the absorption at 280 nm. Note that if the concentration of the loaded protein solution is rather high, the band corresponding to a hFRB dimer (Fig. 1, lanes 8–9) becomes accordingly stronger. Refolding of hFRB Refolding of hFRB was achieved by drop-wise dilution (1:200) of a stock solution of denatured protein into a native buffer [22]. The denatured protein stock solution was prepared by dissolving the lyophilized protein into 20 mM Tris, 6 M GdmCl, 10 mM TCEP, pH 8 to a final concentration of 0.1–0.9 mM. The dissolved protein was centrifuged 10 min at 13,000 rpm before use. The refolding buffer was always 20 mM Tris, 1 mM TCEP, pH 8 with different supplements (150 or 300 mM NaCl or 50 mM l-Glu/50 mM l-Arg or 20 mM l-Leu or 30% propanol-2). The buffer containing additionally 300 mM NaCl was the final refolding buffer. Refolding on a small scale (0.5 ml denatured protein solution, 100 ml refolding buffer) was done by injecting the denatured protein solution in small steps (7–10 within about 5–8 h, each 0.05–0.07 ml) from a 1 ml syringe with needle into a screw cap bottle on a magnetic stirrer that was kept at 4 °C. After all the denatured protein had been injected into the refolding buffer, the solution kept stirring overnight at 4 °C. The diluted protein solution was concentrated using an ultrafiltration spin column (Vivaspin20 from Vivascience) with an MWCO of 5000 according to the manufacturer’s manual. Refolding on a large scale was done by gravity flow from a syringe with a fine needle (BD Microlance™ 3, 25 G × 5/8′′, 0.5 mm × 16 mm). Refolding on an even larger scale (>20 ml) may be done using an electronic infusion injector or a peristaltic pump. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |