RESUMEN
A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The alpha-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone alpha-subunit. The beta-subunit DNA construct for C3 encodes residues 1-145 from human chorionic gonadotropin (hCG)-beta with the exceptions that FSH beta amino acid 88 (D) is substituted for hCG beta amino acid 94 (R) and FSH beta amino acids 95-108 (TVRGLGPSYCSFGE) are substituted for hCG beta amino acids 101-114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.
Asunto(s)
Gonadotropina Coriónica/genética , Hormona Folículo Estimulante/genética , Proteínas Recombinantes de Fusión/farmacología , Secuencia de Aminoácidos , Animales , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica Humana de Subunidad beta/química , Gonadotropina Coriónica Humana de Subunidad beta/genética , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/fisiología , Femenino , Hormona Folículo Estimulante/farmacología , Hormona Folículo Estimulante de Subunidad beta/química , Hormona Folículo Estimulante de Subunidad beta/genética , Humanos , Datos de Secuencia Molecular , Tamaño de los Órganos/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Ratas , Receptores de HFE/metabolismo , Receptores de HL/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Útero/anatomía & histologíaRESUMEN
The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-alpha. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation.