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Expression and characterization of biologically active ovine FSH from mammalian cell lines
P S Mountford1, M R Brandon, T E Adams
1Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Abstract:
Stably transfected cell lines expressing the alpha subunit, beta subunit and alpha/beta heterodimer of ovine (o)FSH have been established following the transfection of Chinese hamster ovary cells with alpha and beta subunit cDNA expression vectors. In the absence of the alpha subunit, FSH beta subunit polypeptides were inefficiently secreted and displayed a short intracellular half-life, while free alpha subunits were readily secreted in the absence of the beta subunit. Cotransfection of oFSH alpha and beta subunit cDNAs led to heterodimer assembly and secretion. While alteration of the nucleotide sequence flanking the beta subunit AUG initiation codon did not appreciably enhance heterodimer biosynthesis and secretion, the replacement of the 5' untranslated and signal peptide-coding regions of the beta subunit cDNA with the corresponding sequences from an oGH cDNA clone was associated with a twofold increase in oFSH heterodimer secretion. The recombinant oFSH had a higher molecular weight than pituitary-derived oFSH, and was more acidic than the native hormone when analysed using isoelectric focusing, suggesting a greater degree of sialylation of the recombinant hormone. A comparison of the activities of the recombinant and native hormones in the porcine testis radioreceptor assay and in the in vitro Sertoli cell bioassay revealed that the recombinant oFSH displayed enhanced biological activity in the Sertoli cell assay when compared with the native hormone.
Insights
Researchers created recombinant ovine follicle-stimulating hormone (oFSH) by expressing its alpha and beta subunits in Chinese hamster ovary cells. This engineered oFSH demonstrated enhanced biological activity in Sertoli cell assays compared to native oFSH.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Follicle-stimulating hormone (FSH) is crucial for reproductive processes.
- Efficient production of functional FSH requires proper assembly of its alpha and beta subunits.
- Understanding subunit interactions is key to producing recombinant FSH.
Purpose of the Study:
- To establish stably transfected cell lines for producing ovine FSH (oFSH) alpha and beta subunits and their heterodimer.
- To investigate the impact of specific cDNA modifications on oFSH heterodimer biosynthesis and secretion.
- To compare the biological activity of recombinant oFSH with native pituitary-derived oFSH.
Main Methods:
- Transfection of Chinese hamster ovary (CHO) cells with oFSH alpha and beta subunit cDNA expression vectors.
- Engineering of the beta subunit cDNA by modifying flanking sequences and signal peptide regions.
- Analysis of recombinant oFSH molecular weight, charge (isoelectric focusing), and sialylation.
- Assessment of biological activity using a porcine testis radioreceptor assay and an in vitro Sertoli cell bioassay.
Main Results:
- Free alpha subunits were secreted, while free beta subunits were not, indicating the need for heterodimerization.
- Cotransfection led to oFSH heterodimer assembly and secretion.
- Modifying the 5' untranslated and signal peptide regions of the beta subunit cDNA increased heterodimer secretion twofold.
- Recombinant oFSH exhibited higher molecular weight, greater acidity (increased sialylation), and enhanced biological activity in the Sertoli cell assay compared to native oFSH.
Conclusions:
- Successful production of recombinant oFSH (oFSH) via stable transfection of CHO cells.
- Specific genetic modifications significantly enhance the secretion of functional oFSH heterodimers.
- Recombinant oFSH displays superior biological activity, suggesting potential therapeutic advantages over native hormone.