Refolding Record:
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 3 |
| Molecular Weight | 203.2 |
| Pi | 5.52 |
| Molecular Weight | 203.2 |
| Disulphides | Unknown |
| Full Sequence |
n/a
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | McCusker E, Robinson AS. (2008) Protein Expression and Purification, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET15b |
| Expression Protocol | E. coli BL21 (DE3) cells harboring the pET15b-H6-Gα constructs were grown in 1.5 L enriched media (2% tryptone, 1% yeast extract, 0.5% NaCl, 0.2% glycerol, and 50 mM KH2PO4, pH 7.2) in the presence of 50 μg/ml ampicillin at 37 °C to an OD600 of 0.6 in baffled shaker flasks and then induced with 1 mM IPTG for 4 h (multiple conditions were tested and under all conditions the Gα subunits formed inclusion bodies). Cell cultures were harvested by centrifugation at 8000 rpm for 15 min at 4 °C. At the time of harvest, OD600 was 2.0 for the Gα subunits expressing cells. Cell pellets were rapidly frozen in liquid nitrogen and stored at −80 °C. Cell pellets were resuspended to 1/10 culture volume (150 ml) in buffer containing 50 mM Tris–HCl (pH 8.0), 20 mM β-mercaptoethanol (βMe), and 8 M urea. To facilitate lysis, the cell lysate was sonicated on ice for 15 min at 30% duty cycle with 30% output using a Branson 450 sonicator. The lysate was incubated with pre-equilibrated Ni–NTA (Qiagen) resin. The column was washed with 20 mM Tris–HCl (pH 8.0), 20 mM βMe, 500 mM NaCl, 8 M urea, and 40 mM imidazole. The Gα subunits were eluted with 20 mM Tris–HCl (pH 8.0), 20 mM βMe, 300 mM NaCl, 8 M urea, 10% glycerol, with a series of increasing step gradients of imidazole in the range of 60–250 mM. The eluted protein fractions were stored at room temperature. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris–HCl (pH 8.0), 20 mM β-mercaptoethanol (βMe), and 8 M urea |
| Solubilization Buffer | 20 mM Tris–HCl (pH 8.0), 20 mM βMe, 500 mM NaCl, 8 M urea, and 40 mM imidazole |
| Refolding Buffer | 20 mM Tris–HCl, pH 8.0, 20% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1% CHAPS, 100 μM GDP |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | A rapid dilution technique was employed to efficiently refold the Gα subunits. The Gα subunits were diluted 20-fold (<10 μg/ml) into refolding buffer (20 mM Tris–HCl, pH 8.0, 20% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1% CHAPS, 100 μM GDP) at room temperature. The Gα subunits were added dropwise (1 drop per 20 s) into refolding buffer under rapid stirring at room temperature, then incubated 16–72 h at 4 °C with gentle stirring. Amicon Ultra (Millipore 30,000 molecular weight cut-off) device was used to concentrate the sample. Samples were dialyzed against 1 L refolding buffer at 4 °C, without GDP, to remove excess unbound GDP. Gα subunits were passed through a Millex syringe driven 0.22 μm filter unit (Millipore) to eliminate any large aggregates that may have formed in the refolding process. |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | CHAPS |
| Additives Concentration | 0.1% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |