Available online 7 May 2024

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The classic way to produce single-chain (sc) glycoprotein hormones is to fuse their two subunits through the carboxy-terminal peptide (CTP) from human Choriogonadotropin (hCG). The CTP confers a longer half-life to single-chain hormones thanks to its four O-glycosyl side chains. However, unlike syncytiotrophoblastic cells, most cells used for recombinant protein production do not transfer O-glycosyl chains efficiently. We thus choose to fuse the hFSH subunits with a linker comprising two N-glycosyl side chains (sc-hFSH LNN) or none (sc-hFSH L0N), that were generated using two expression systems, HEK293 and CHO K1 cells. Their production levels and biological activities were tested and compared. Both expression systems successfully produced biologically active sc-hFSH, but, in our hands, CHO K1 cells yielded about 30-fold higher amounts of recombinant protein than HEK293 cells. Moreover, sc-hFSH L0N was considerably less expressed than sc-hFSH LNN in both cell types. Our data show that sc-hFSH L0N and sc-hFSH LNN produced from both cell lines stimulate cAMP and progesterone production in mLTC cells expressing hFSH receptors and exhibit similar B/I (in vitro Bioactivity / Immuno activity) ratios. Finally, the ratio of in vivo / in vitro bioactivities for sc-hFSH LNN relative to natural pituitary heterodimeric hFSH increased 8-fold, most likely because of a longer half-life in the blood.

Section snippetsINTRODUCTION

Human Follicle-Stimulating Hormone (hFSH), like Luteinizing Hormone (hLH), is a pituitary gonadotropin targeting the gonadal cells of both men and women. Structurally, gonadotropins are heterodimeric glycoprotein hormones, also including pituitary Thyroid-Stimulating Hormone (TSH). Moreover, a Chorionic Gonadotropin (hCG) also exists in the human species, secreted by the placenta during gestation. All these hormones consist of a common α-subunit, non-covalently linked to different

Chemicals and reagents

All chemicals were purchased from Sigma–Aldrich unless otherwise noted. Tris/glycine buffer (10X) and Low-Range SDS-PAGE Standards (Catalog # 1610304) were obtained from Bio-Rad (Hercules, CA). The secondary antibody anti-rabbit IgG (H+L) (CFTM770 conjugated antibodies) was purchased from Biotium (Hayward, CA); pGlosensor–TM -22F cyclic AMP plasmid was from Promega (France), XtremeGENE HP DNA transfection reagent was from Roche (France). Precision Plus Protein Standards Dual Color was obtained

Expression of single-chain hFSHs in HEK293 cells and CHO K1 cells

The pcDNA3 single-chain hFSHβ-LNN-α (sc-hFSH LNN) and hFSHβ-L0N-α (sc-hFSH L0N) were transfected in HEK293 cells and CHO K1 cells in serum-free medium. After 7 days of incubation, the media were collected and assayed by ELISA.

The quantities of recombinant hormones secreted by HEK293 cells and CHO K1 cells were estimated in a sandwich ELISA using standard hFSH SIAFP (10μg/μl) (Fig. 2). The productions of sc-hFSH LNN and sc-hFSH L0N were 1.628 ± 0.137ng/μl and 0.108 ± 0.050ng/μl respectively in

DISCUSSION

The β and α subunits were fused via very similar 23aa-peptide sequences differing only by two Ala/Thr replacements and therefore including or not two N-glycosyl sites (Fig. 1). The recombinant sc-hFSH LNN and sc-hFSH L0N proteins were then successfully produced using two different expression systems, HEK293 and CHO K1 cell lines. The expression yield was found to be about 30-fold higher in CHO K1 cells than in HEK293 cells and, in both cell lines, better for sc-hFSH LNN than for sc-hFSH L0N. In

Funding

Research is supported by Vingroup Innovation Foundation (VINIF) in project code VINIF.2020.DA05. It is also supported by GIS FC3R (22FC3R-008) from the fund managed by Inserm (France) for TMDN internship in France.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

The authors are grateful to Dr Danièle KLETT (INRAE, CNRS, Nouzilly, France) for fruitful discussions.

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