Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glycine receptor alpha-1 chain precursor |
| Abbreviated Name | GlyR |
| SCOP Family | Transmembrane helical fragments |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P23415 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain (29-248) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 256 |
| Molecular Weight | 29392.4 |
| Pi | 6.41 |
| Molecular Weight | 29392.4 |
| Disulphides | 2 |
| Full Sequence |
MHHHHHHSSGLVPRGSGMKETAAAKPGRQHMDSPDLARSATKPMSPSDFLDKLMGRTSGYDARIRPNFKGPPVNVSCNIFINSFGSIAETTMDYRVNIFLRQQWNDPRLAYNEYPDDSLDLDPSMLDSIWKPDLFFANEKGAHFHEITTDNKLLRISRNGNVLYSIRITLTLACPMDLKNFPMDVQTCIMQLESFGYTMNDLIFEWQEQGAVQVADGLTLPQFILKEEKDLRYCTKHYNTGKFTCIEARFHLERQM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Breitinger, U., Breitinger, H. G., Bauer, F., Fahmy, K., Glockenhammer, D., Becker, C. M. (2004) J Biol Chem, 279, 1627-1636 |
| Project Aim | Structure-Function |
| 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 | not stated |
| Expression Vector | pET-30a |
| Expression Protocol | Cells were harvested after induction and resuspended in 50mM TrisHCl, 2.5 mM EDTA, pH 7.4 and treated with lysozyme,(0.1mg/ml, 30min, 0degC). The cells were then sonicated. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM TrisHCl, 2.5 mM EDTA, 0.5% Triton X-100, pH 8.0 |
| Solubilization Buffer | 8M urea, 100mM NApi, 10mM TrisHCl, pH 8.0 |
| Refolding Buffer | 20mM TrisHCl, pH 8.0 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were washed 4x with washing buffer and dissolved in solubilisation buffer. The denatured protein was then applied to a Ni-agrose column, purified and eluted with increasing imidazole concentrations. Refolding was preformed 2h after denaturing with 10microL of dissolved inclusion bodies being added to 990microL dilution solution (55 mM Tris–HCl, pH 8.2, 264 mM NaCl, 1 mM DTT, 0.055% (w/v) PEG 4000). Samples were incubated overnight at 4degC before dialysis for 6h against refolding buffer. |
| Refolding Assay | Structure Determination |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |