Refolding Record:
| Protein | |
|---|---|
| Protein Name | Acetylcholine Receptor alpha-Subunit |
| Abbreviated Name | AChR |
| SCOP Family | Neurotransmitter-gated ion-channel transmembrane pore |
| Structure Notes | |
| Organism | Torpedo marmorata |
| UniProt Accession | P02711 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | N-terminal extracellular domain aa.1-209 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 212 |
| Molecular Weight | 24930.5 |
| Pi | 5.55885 |
| Molecular Weight | 24930.5 |
| Disulphides | 2 |
| Full Sequence |
SEHETR LVANLLENYN KVIRPVEHHT HFVDITVGLQ LIQLINVDEV NQIVETNVRL RQQWIDVRLR WNPADYGGIK KIRLPSDDVW LPDLVLYNNA DGDFAIVHMT KLLLDYTGKI MWTPPAIFKS YCEIIVTHFP FDQQNCTMKL GIWTYDGTKV SISPESDRPD LSTFMESGEW VMKDYRGWKH WVYYTCCPDT PYLDITYHFI MQR SGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Schrattenholz A, Pfeiffer S, Pejovic V, Rudolph R, Godovac-Zimmermann J, Maelicke A (1998) J Biol Chem, 273, 32393-9 |
| Project Aim | Undefined,Structure-Function,Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pET3a |
| Expression Protocol | no details provided |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 3M guanidinium chloride, 10mM Tris-HCl, 100mM NaCl, pH 8.4 |
| Solubilization Buffer | 6M guanidinium chloride, 100mM Tris-HCl, 100mM dithioerythritol, pH 8.5 |
| Refolding Buffer | 100mM Tris-HCl, 5mM GSH, 0.5mM GSSG, 10mM EDTA, 1M L-arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.5 |
| Refolding Temperature | 10.0 |
| Protein Concentration | |
| Refolding Time | 24h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5mM/0.5mM |
| Refolding Protocol | The pellet containing the nAChR1-209 fragment was solubilized in guanidinium chloride (6 M), Tris-HCl (0.1 M), dithioerythritol (0.1 M) (pH 8.5) by ultrasonic treatment and incubation for 2 h at 20degC. In order to remove dithioerythritol, the mixture was dialyzed in a hollow-fiber membrane module (Variperm L, BITOP, Witten, Germany) for 24 h against a 50-fold volume of Tris-HCl (0.1 M), EDTA (0.01 M), guanidinium chloride (6 M), pH 8.5. For a typical renaturation batch, 100microl of this dialysate were diluted in 20 ml of renaturation buffer, consisting of Tris-HCl (100 M), glutathione (reduced form) (5 M), glutathione (oxidized form) (0.5 mM), EDTA (10 mM), arginine (1000 mM), pH 9.5, and incubated for 24 h at 10degC. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 1M |
| Refolding Yield | |
| Purity | 50% |
| Notes | |