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 |