التفاصيل البيبلوغرافية
| العنوان: |
Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells. |
| المؤلفون: |
Byambaragchaa, Munkhzaya1,2 (AUTHOR) munkhzaya@hknu.ac.kr, Park, Sei Hyen3 (AUTHOR) mrtree119@naver.com, Kim, Sang-Gwon3 (AUTHOR) tom0391@naver.com, Shin, Min Gyu4 (AUTHOR) smg159@korea.kr, Kim, Shin-Kwon4 (AUTHOR) ksk4116@korea.kr, Park, Myung-Hum5 (AUTHOR) pmh@tntresearch.co.kr, Kang, Myung-Hwa6 (AUTHOR) mhkang@hoseo.edu, Min, Kwan-Sik1,2,7 (AUTHOR) ksmin@hknu.ac.kr |
| المصدر: |
International Journal of Molecular Sciences. Jul2024, Vol. 25 Issue 13, p7282. 18p. |
| مصطلحات موضوعية: |
*FOLLICLE-stimulating hormone, *EELS, *PEPTIDES, *CHO cell, *MOLECULAR weights, *AMINO acids |
| مستخلص: |
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 N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 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. [ABSTRACT FROM AUTHOR] |
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