Byambaragchaa, M.; Park, S.H.; Kim, S.-G.; Shin, M.G.; Kim, S.-K.; Hur, S.-P.; Park, M.-H.; Kang, M.-H.; Min, K.-S. Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells. Curr. Issues Mol. Biol.2024, 46, 6085-6099.
Byambaragchaa, M.; Park, S.H.; Kim, S.-G.; Shin, M.G.; Kim, S.-K.; Hur, S.-P.; Park, M.-H.; Kang, M.-H.; Min, K.-S. Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells. Curr. Issues Mol. Biol. 2024, 46, 6085-6099.
Byambaragchaa, M.; Park, S.H.; Kim, S.-G.; Shin, M.G.; Kim, S.-K.; Hur, S.-P.; Park, M.-H.; Kang, M.-H.; Min, K.-S. Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells. Curr. Issues Mol. Biol.2024, 46, 6085-6099.
Byambaragchaa, M.; Park, S.H.; Kim, S.-G.; Shin, M.G.; Kim, S.-K.; Hur, S.-P.; Park, M.-H.; Kang, M.-H.; Min, K.-S. Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells. Curr. Issues Mol. Biol. 2024, 46, 6085-6099.
Abstract
We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eCG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the eel LH-wt and eel LH-M plasmids were isolated from five and nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5,000–7,500 ng/mL on day 9. The molecular weight of eel LH-wt was 32–36 kDa while that of eel LH-M increased to approximately 38–44 kDa, indicating the detection of two bands. Treatment with peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new rec-eel LH analog which contains the eCG β-subunit CTP linker comprising O-linked glycosylation sites, had more potent activity than the wild-type in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.
Keywords
Eel LH-M; Stable expression; CHO DG44 cells; cAMP response; pERK1/2
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.