High-level expression of a functional single-chain human chorionic gonadotropin-luteinizing hormone receptor ectodomain complex in insect cells.
Reproductive capacity in primates is dependent on the high-affinity binding of the glycoprotein hormones LH and human (h)CG to the large ectodomain (ECD) of their common receptor (LHR). Our understanding of the precise molecular determinants of hormone binding is limited, because there are no structural data for any of the glycoprotein hormone receptors. Overexpression of the ECD of the receptor has been attempted in various expression systems. Prokaryotic expression does not yield properly folded ECD. Eukaryotic expression, on the other hand, results in mostly heterogeneous, intracellularly trapped protein, but the secreted ECD is completely folded. Accordingly, we have tethered the single-chain hormone, yoked hCG, to the N terminus of LHR-ECD (yoked hormone-extracellular domain). Yoked hCG is secreted at high levels; binds LHR with high affinity; and, when tethered to the N terminus of full-length LHR, it binds and constitutively activates the receptor. Using recombinant baculovirus, yoked hormone-extracellular domain is secreted from insect cells at levels greater than 1 microg/ml, nearly 20-fold higher than that previously reported in eukaryotic expression systems. The protein was purified and binds exogenous (125)I-hCG with high affinity but, significantly, only after protease treatment to remove the tethered hormone. Thus, the fusion protein seems to form a functional hormone-receptor complex that is expressed at levels sufficient for its biophysical characterization.
Fralish
G B
GB
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, USA.
Narayan
P
P
Puett
D
D
D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D007313:Insecta / Q000378:metabolism*; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface / Q000378:metabolism; D011974:Receptors, LH / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin
C107489:BIRC5 protein, human; D006063:Chorionic Gonadotropin; D000939:Epitopes; D051034:Inhibitor of Apoptosis Proteins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D011994:Recombinant Proteins; D000077022:Survivin