Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) beta-subunit, was attached between the beta- and alpha-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LH beta/alpha-wt and FSH beta/alpha-wt proteins. The molecular weight of eel LH beta/alpha-wt and eel FSH beta/alpha-wt was 32-34 and 34-36 kDa, respectively, and that of LH-M and FSH-M was 40-43 and 42-45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8-10 kDa. The EC50 value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.