Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.
The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that alpha211, the entire amino-terminal extracellular domain of AChR alpha subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The alpha211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mm ( approximately 30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with alpha-bungarotoxin (k(D) 0.2 nm) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of alpha211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently (15)N-labeled in P. pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.
Yao
Yun
Y
Department of Neurobiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, PA 15261, USA.
Wang
Junmei
J
Viroonchatapan
Nitnara
N
Samson
Avraham
A
Chill
Jordan
J
Rothe
Elizabeth
E
Anglister
Jacob
J
Wang
Zuo-Zhong
ZZ
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014101:Torpedo; D014961:X-Ray Diffraction
D018076:DNA, Complementary; D008024:Ligands; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins