Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells
Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG4...
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MDPI AG
2025-02-01
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| author | Munkhzaya Byambaragchaa Sei Hyen Park Myung-Hum Park Myung-Hwa Kang Kwan-Sik Min |
| author_facet | Munkhzaya Byambaragchaa Sei Hyen Park Myung-Hum Park Myung-Hwa Kang Kwan-Sik Min |
| author_sort | Munkhzaya Byambaragchaa |
| collection | DOAJ |
| description | Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter CHO-K1 cells. The expression from one, among nine isolates, peaked at 364–470 IU/mL on days 9 and 11. The molecular weight of rec-eCG β/α ranged from 40 to 47 kDa, with two distinct bands. PNGase F treatment reduced the molecular weight by 8–10 kDa, indicating N-glycosylation. Rec-eCG β/α demonstrated dose-responsive cAMP activity in cells expressing eLH/CGR, with enhanced potency in rLH/CGR and rFSHR. Phospho-ERK1/2 activation peaked at 5 min before declining rapidly. β-arrestin 2 recruitment was receptor-mediated in cells expressing hFSHR and hLH/CGR. This study provides insights into the mechanisms underlying eCG’s FSH- and LH-like activities. Stable CHO DG44 cells can produce large quantities of rec-eCG. eCG activates pERK1/2 signaling via the PKA/cAMP pathway and facilitates β-arrestin 2 recruitment. |
| format | Article |
| id | doaj-art-87b0761a9d114c4ab2d7a90a8a7089eb |
| institution | DOAJ |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj-art-87b0761a9d114c4ab2d7a90a8a7089eb2025-08-20T02:44:31ZengMDPI AGBiomolecules2218-273X2025-02-0115228910.3390/biom15020289Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 CellsMunkhzaya Byambaragchaa0Sei Hyen Park1Myung-Hum Park2Myung-Hwa Kang3Kwan-Sik Min4Carbon-Neutral Resources Research Center, Hankyong National University, Anseong 17579, Republic of KoreaGraduate School of Animal Biosciences, Hankyong National University, Anseong 17579, Republic of KoreaTNT Research, Sejong 30141, Republic of KoreaDepartment of Food Science and Nutrition, Hoseo University, Asan 31499, Republic of KoreaCarbon-Neutral Resources Research Center, Hankyong National University, Anseong 17579, Republic of KoreaEquine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter CHO-K1 cells. The expression from one, among nine isolates, peaked at 364–470 IU/mL on days 9 and 11. The molecular weight of rec-eCG β/α ranged from 40 to 47 kDa, with two distinct bands. PNGase F treatment reduced the molecular weight by 8–10 kDa, indicating N-glycosylation. Rec-eCG β/α demonstrated dose-responsive cAMP activity in cells expressing eLH/CGR, with enhanced potency in rLH/CGR and rFSHR. Phospho-ERK1/2 activation peaked at 5 min before declining rapidly. β-arrestin 2 recruitment was receptor-mediated in cells expressing hFSHR and hLH/CGR. This study provides insights into the mechanisms underlying eCG’s FSH- and LH-like activities. Stable CHO DG44 cells can produce large quantities of rec-eCG. eCG activates pERK1/2 signaling via the PKA/cAMP pathway and facilitates β-arrestin 2 recruitment.https://www.mdpi.com/2218-273X/15/2/289recombinant eCGcAMP signalingCHO-DG44 cellsphospho-ERK1/2β-arrestin 2 recruitment |
| spellingShingle | Munkhzaya Byambaragchaa Sei Hyen Park Myung-Hum Park Myung-Hwa Kang Kwan-Sik Min Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells Biomolecules recombinant eCG cAMP signaling CHO-DG44 cells phospho-ERK1/2 β-arrestin 2 recruitment |
| title | Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells |
| title_full | Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells |
| title_fullStr | Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells |
| title_full_unstemmed | Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells |
| title_short | Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells |
| title_sort | enhanced production and functional characterization of recombinant equine chorionic gonadotropin rec ecg in cho dg44 cells |
| topic | recombinant eCG cAMP signaling CHO-DG44 cells phospho-ERK1/2 β-arrestin 2 recruitment |
| url | https://www.mdpi.com/2218-273X/15/2/289 |
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