Expression of a recombinant bifunctional protein from a chimera of human lutropin receptor and human chorionic gonadotropin beta-subunit.
Human lutropin (hLH) and human chorionic gonadotropin (hCG) are structurally and functionally similar and play important roles in reproduction via a common gonadal receptor (LH-R). However, hormone specific hCG-beta subunit contains 24 additional amino acid carboxyl terminal peptide (CTP), which produce specific antibodies to hCG-beta with little cross-reaction with LH. A chimeric protein containing both hLH-R and hCG-beta would provide a unique bifunctional antigen for immunocontraception. In this study is described the synthesis of a chimeric DNA construct of full-length of hLH-receptor and hCG-beta and its expression in Sf9 cells to produce a bifunctional protein. Recombinant protein was recognized by antibodies to LH-R as well as anti-hCG-beta in Western Blots, thus indicating the preservation of immunological epitopes for both hLH-R and hCG-beta in the chimera. Specific ligand binding of recombinant hLH-R component was demonstrated by the displacement of bound labeled hCG at increasing concentrations of unlabeled hCG, indicating that, the presence of hCG-beta component of the chimera did not interfere with the binding of hCG to LH-R. hCG-beta was also present in the recombinant chimeric protein as shown by a specific hCG-beta chemiluminescence assay. Treatment of transfected Sf9 cells with hCG induced dose-dependent increase in the stimulation of intracellular cAMP production, which showed that the ligand binding had functional activity. These results demonstrate that the chimeric DNA construct of hLHR-hCG-beta expressed a bifunctional protein containing both hLH-R and hCG-beta activities, which could provide a unique potential antigen for immunocontraception in vertebrates.
Hao
Meirong
M
Division of Reproductive Endocrinology, Department of OB-GYN, Weill Medical College of Cornell University, New York, NY 10021, USA.
Rathnam
P
P
Saxena
Brij
B
D000818:Animals; D002460:Cell Line; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D018997:Chorionic Gonadotropin, beta Subunit, Human; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP