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KMID : 0613820170270080864
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Journal of Life Science
2017 Volume.27 No. 8 p.864 ~ p.872
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Biochemical Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG), Using CHO Cells and PathHunter Parental Cells Expressing Equine Luteinizing Hormone/Chorionic Gonadotropin Receptors (eLH/CGR)
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Lee So-Yun
Byambaragchaa Munkhzaya
Kim Jeong-Soo
Seong Hun-Ki
Kang Myung-Hwa
Min Kwan-Sik
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Abstract
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Equine chorionic gonadotropin (eCG) consists of highly glycosylated α- and β-subunits and is a unique member of the gonadotropin family, because it elicits the response characteristics of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. To directly assess the biological function of rec-eCGβ/α, we constructed mammalian expressing vectors of equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). The activity of rec-eCGβ/α in vitro assayed in transient transfected CHO-K1 cells and in stably transfected PathHunter Parental cells with eLH/CGR was investigated. rec-eCGβ/α was efficiently secreted in the CHO-K1 suspension cell media, and the quantity detected was about 200 mIU/ml from 1 to 7 days after transfection. In the western blot analysis, the rec-eCGβ/α protein was broadly identified to be about 40~45 kDa molecular weight. The cAMP stimulation in CHO-K1 cells expressing eLH/CGR was determined to evaluate the activity of rec-eCGβ/α. The cAMP concentration increased in direct proportion to the concentration of the rec-eCGβ/α. The EC50 value in the transient transfected CHO-K1 cells was 8.1±6.5 ng. The stable cell lines of eLH/CGR were established in the PathHunter Parental cells expressing β-arrestin. We found that rec-eCGβ/α had full LH activity in the PathHunter Parental cells expressing eLH/CGR. The EC50 value in transient and stable cells was 5.0±4.7 ng/ml and 4.5±5.2 ng/ml, respectively. These results suggest that rec-eCGβ/α has a biological activity in a cell expressing eLH/CGR. These stable cells expressed in PathHunter Parental cells could be useful for elucidating the functional mechanisms of deglycosylated rec-eCGβ/α mutants.
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KEYWORD
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cAMP, CHO cells, eLH/CGR, PathHunter Parental cells, rec-eCGβ/α
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