Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor α-subunit

• Published in: Gene Vol. 98; no. 2; pp. 289 - 293
• Main Authors: Talib, Sohel, Leiby, Kevin R., Wright, Kathy, Okarma, Thomas B.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.02.1991; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Biological and medical sciences; Bungarotoxins - metabolism; Cloning, Molecular - methods; coupled transcription-translation; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Synthetic; Humans; Macromolecular Substances; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Muscles - physiology; myasthenia gravis; Myasthenia Gravis - immunology; Plasmids; Protein Biosynthesis; Receptors, Cholinergic - genetics; Receptors, Cholinergic - immunology; Receptors, Cholinergic - metabolism; Recombinant DNA; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Sequence Homology, Nucleic Acid; Transcription, Genetic; α-bungarotoxin binding; aa; SDS; nt; myasthenia gravis; PAGE; Recombinant DNA; α-bungarotoxin binding; bp; αBgt; AChR; LB; kb; MIR; IPTG; coupled transcription-translation; MG; oligo; Human; Heterologous system; Nervous system diseases; Nucleotide sequence; Escherichia coli; Autoimmune disease; Gene expression; Immunoprecipitation reaction; Cholinergic receptor; Myasthenia gravis; Bacteria; Molecular cloning; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(91)90188-H

To better define the antigenic sites on the human muscle acetylcholine receptor (AChR) that are involved in stimulating the production of pathogenic antibodies in myasthenia gravis (MG), the nucleotide sequence encoding the major extracellular domain of the AChR α subunit was chemically synthesized. The gene cassettes encoding amino acids (aa) 1–85 ( AChR-I) and 86–210 ( AChR-II), were cloned individually, and the coding sequence representing the complete major extracellular domain (aa 1–210; AChR-C)was obtained by subsequent fusion of cassettes encoding AChR-I and AChR-II. The genes were inserted into the inducible expression plasmid, pKK-223-3, and expressed in vitro and in vivo in Escherichia coli. Biological activity was demonstrated by immunoprecipitation of in vitro-synthesized AChR-C by sera from MG patients and by the α-bungarotoxin-binding activity of E. coli-synthesized AChR-II and AChR-C. The availability of the recombinant AChR polypeptides should facilitate studies on the molecular basis of the autoimmune response m MG.