Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor α subunit

Yun Yao, Junmei Wang, Nitnara Viroonchatapan, Avraham Samson, Jordan Chill, Elizabeth Rothe, Jacob Anglister, Zuo Zhong Wang

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


The a subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for α-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 α 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 α211, the entire amino-terminal extracellular domain of AChR α subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The α211 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 (∼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 α-bungarotoxin (kD 0.2 nM) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of α211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently 15N-labeled in P. pastoris, and proton crosspeaks 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.

Original languageEnglish
Pages (from-to)12613-12621
Number of pages9
JournalJournal of Biological Chemistry
Issue number15
StatePublished - 12 Apr 2002
Externally publishedYes


FundersFunder number
National Institute of Neurological Disorders and StrokeR01NS038301


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