Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.
The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson (Rutgers) Medical School, Piscataway 08854.
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Campbell
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Barnett
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Moyle
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011974:Receptors, LH / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D015292:Glycoprotein Hormones, alpha Subunit; D011974:Receptors, LH; D011994:Recombinant Proteins