Physical chemical, immunological and biological characterization of arapaima 2 gigas follicle-stimulating hormone (ag-FSH) synthesized in HEK293 cells

Abstract

The cDNA sequences of the common gonadotrophic hormone α‐subunit (ag‐GTHα) and of the specific follicle‐stimulating hormone β‐subunit (ag‐FSHβ) of the giant Amazonian fish Arapaima gigas, have been previously isolated by our research group. A synthesis of ag‐FSH in HEK293 cells and its purification and preliminary characterization were also carried out. In the present work, ag‐FSH was obtained and purified from the same host cells and an extensive physical chemical characterization was performed via RP‐HPLC, HPSEC and MALDI‐TOF‐MS. Ag‐FSH, when compared to human FSH (hFSH), showed a higher hydrophobicity by RP‐HPLC and a higher molecular mass (MM) via HPSEC. The same higher MM was also confirmed via MALDI‐TOF‐MS: 35,353 Da for ag‐FSH against 31,969 Da for hFSH. Its immunological activity was also confirmed via an hFSH ELISA, in comparison with the highly purified pituitary preparation of hFSH‐AFP7298A, from the National Hormone and Pituitary Program (NHPP‐USA). Finally, an in‐vitro bioassay, based on the stimulated release of 11‐ketotestosterone (11‐KT) from immature A. gigas testis, quantified its biological activity in comparison with human chorionic gonadotropin (hCG) and with the WHO International Standard of recombinant hFSH (NIBSC‐08/282) This suggests that injections of ag‐FSH in A. gigas and ag‐FSH cDNA Gene Therapy applications should improve the reproductive functions of this threatened species.