Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor alpha-subunit.
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 alpha 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 alpha-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 in MG.
Talib
S
S
Applied Immune Sciences Inc., Menlo Park, CA 94025-1109.
Leiby
K R
KR
Wright
K
K
Okarma
T B
TB
D000595:Amino Acid Sequence; D002038:Bungarotoxins / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005813:Genes, Synthetic; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology*; D009157:Myasthenia Gravis / Q000276:immunology; D010957:Plasmids; D014176:Protein Biosynthesis; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D002038:Bungarotoxins; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins