Single chain human chorionic gonadotropin, hCGαβ: effects of mutations in the α subunit on structure and bioactivity

摘要: The strategy of translationally fusing the subunits heterodimeric proteins into single chain molecules is often used to overcome mutagenesis-induced defects in subunit interactions. approach α and β human Chorionic Gonadotropin (hCG) produce a hormone (phCGαβ) was investigate roles critical residues receptor interaction biological activity. mutated using PCR-based site-directed mutagenesis, fused wild type fusion protein expressed Pichia pastoris expression system. Following partial purification, mutant were extensively characterized immunological probes, assays, vitro bioassays. mutation hCGα P38A, which disrupts molecule, produced molecule exhibiting altered interactions as judged by criteria, but could bind with lower affinity elicit response. Mutation T54A disrupting glycosylation at Asparagine 52, believed be important for bioactivity, also yielded biologically active suggesting that this site not bioactivity it case heterodimer. generate superagonist action. Introduction four lysine Loop 1 led generation having higher enhanced bioactivity. Immunological characterization revealed between identical those seen hormone, appeared retain their isolated conformations, retained ability receptors These data suggest plasticity hormone-receptor