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

Abstract

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) β-subunit, was attached between the β- and α-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 LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-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.

Development of a biotechnology process for the production of a novel hyperglycosylated long‐acting recombinant bovine follicle‐stimulating hormone

“…Through lentiviral transgenesis and a high cell density perfusion process, almost 140 mg of each hormone were produced in 1-L bioreactor in 18 days, with harvests averaging 8-9 µg.mL −1 . These values are higher than those previously reported for the production of FSH in CHO cells (0.2-0.6 µg.mL −1, [26,33] ) and in Leishmania tarentolae (L.…”

“…[8,[11][12][13][14] For these reasons, the development of a recombinant bFSH (rbFSH) represents an attractive alternative to overcome these disadvantages. Despite the production of several nonhuman recombinant, FSH has been reported in bacteria, [15] yeast, [16][17][18][19][20] plant cells, [21] insect cells, [22][23][24] protozoa, [25] and mammalian cells, [26][27][28][29][30][31][32] up to date, animal-derived products are still being used.…”

“…FSH from different species has been successfully modified by the fusion of peptides containing new N-glycosylation sites [28,33,34] as well as by the addition of the carboxyl-terminal peptide (CTP) of the human CG (hCTP) [30,35] or equine CG (eCTP). [26] Through these strategies, it was possible to obtain long-lasting FSH molecules exhibiting a prolonged half-life and increased in vivo biological activity.…”

Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells

“…Almost the same results are obtained in Drosophila S2 cells [ 22 ] and Pichia pastoris [ 23 , 24 ]. These glycoproteins can be produced in Chinese hamster ovary (CHO)-K1 and CHO-suspension (CHO-S) cells; however, they cannot be produced on a large scale [ 2 , 18 , 19 , 25 , 26 ].…”

“…Owing to glycoprotein hormones, such as hCG, FSH, LH, and TSH, the CG β-subunit carboxy-terminal peptide (CTP) linker was found to induce substantially increased in vivo potency and circulatory half-lives [ 27 , 28 , 29 , 30 , 31 , 32 ]. Recently, eLH/CG β-subunit CTP linker attachments in tethered eel LH-wt and FSH-wt caused early secretion and signal transduction [ 25 ]. Remarkable results have been obtained in clinical trials with the hGH-MOD-4023 molecule, fused to hGH via the hCG β-subunit CTP linker (117–145 aa) [ 33 , 34 , 35 , 36 ].…”