Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells

• Published in: International journal of molecular sciences 2024-07, Vol.25 (13), p.7282
• Main Authors: Byambaragchaa, Munkhzaya, Park, Sei Hyen, Kim, Sang-Gwon, Shin, Min Gyu, Kim, Shin-Kwon, Park, Myung-Hum, Kang, Myung-Hwa, Min, Kwan-Sik
• Format: Article
• Language: English
• Subjects: Animals; Biological activity; cAMP response; CHO Cells; Chorionic Gonadotropin - genetics; Chorionic Gonadotropin - pharmacology; Clinical trials; Cricetulus; Dihydrofolate reductase; Eels - genetics; Follicle Stimulating Hormone - metabolism; Follicle Stimulating Hormone - pharmacology; Follicles; Glycoproteins; Glycosylation; Hormones; Kinases; Molecular weight; pERK1/2 analysis; Phosphorylation; Proteins; rec-eel FSH analog; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Signal transduction; stable expression from CHO DG44 cells
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms25137282

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115-149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.