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We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000–7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32–36 kDa, while that of tethered rec-eel LH-M increased to approximately 38–44 kDa, indicating the detection of two bands. Treatment with the 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 tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

Adaptation of dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time-consuming and labor-intensive process because nonadapted DHFR-deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension-adapted DHFR-deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2-359 μM enhanced cell growth in in-house CDM. Addition of 3.6 μM THF to in-house CDM resulted in a more than 2.5-fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9-fold enhancement of maximum viable cell density. An anchorage- and serum-dependent DHFR-deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in-house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension-adapted DHFR-deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR-deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539-1546, 2016 [ABSTRACT FROM AUTHOR]