Expression of biologically-active heterodimeric bovine follicle-stimulating-hormone in milk of transgenic mice. Proc. Natl. Acad. Sci. USA, 88, 8327-31

Department of Obstetrics and Gynecology, Stanford University, Stanford, California, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/1991; 88(19):8327-31. DOI: 10.1073/pnas.88.19.8327
Source: PubMed


Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two post-translationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine alpha and FSH beta subunits and a modified rat beta-casein gene-based expression system. Lines of bigenic mice expressing both subunits have been generated either by coinjection of the subunit transgenes or by mating mice that acquired and expressed transgenes encoding an individual subunit. Up to 60 international units (15 micrograms) of biologically active FSH per ml was detected in the milk of the bigenic mice. These lines provide a model system for studying the post-transcriptional mechanisms that effect the expression and secretion of this heterodimeric hormone.

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Available from: Jeffrey M Rosen, Sep 29, 2014
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    • "Moreover , oligosaccharides modulated glycoprotein hormone efficacy through an influence on hormone conformation [17]. Biologically active recombinant bFSH has been expressed in different systems [2] [3] [4] [5] [6] [7], which appeared to be hyperglycosylated. However, to date, there is no report about expression of less-glycosylated FSH protein or its subunits and investigation of the effect this isoform on its 1046-5928/$ -see front matter Ó 2009 Elsevier Inc. "
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    ABSTRACT: Bovine follicle-stimulating hormone (bFSH), a pituitary gonadotropin, is a heterodimer hormone that consists of a common alpha-subunit non-covalently associated with the hormone-specific beta-subunit. Unfortunately, expression levels of recombinant bFSH or its subunits are invariably low. We report here the secretory expression of biologically active bFSHalpha and bFSHbeta subunit in the methylotrophic yeast Hansenula polymorpha. A slightly higher level of expression of recombinant bFSH subunits was achieved by using the Saccharomyces cerevisiae-derived calnexin (ScCne1) as a chaperone in engineered H. polymorpha strains. The preliminary data also suggested that bFSH subunits expressed in H. polymorpha appeared to be less-glycosylated. This isoform had been shown to be 80% increase in in vivo bioactivity compared with the hyperglycosylated Pichia pastoris-derived recombinant bFSHalpha/beta. More sophisticated applications of bFSH would profit from the assembled less-glycosylated heterodimer.
    Full-text · Article · Aug 2009 · Protein Expression and Purification
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    • "Post translational modifications , in particular N - glycosylation and sialylation are crucial for the in vivo bioactivity and biostability of FSH ( Morell et al . , 1971 ; Sairam and Bhargavi , 1985 ; Baenziger and Green , 1988 ) . Mammalian cells ( Chappel et al . , 1988 ; Greenberg et al . , 1991 ) were shown to be capable of performing the required post - translational modifications . Therefore they are discussed to be superior to other recombinant production systems including insect ( van de Wiel et al . , 1998 ) , yeast ( Samaddar et al . , 1997 ) , and plant ( Dirnberger et al . , 2001 ) . The mammary gland of trans - genic "
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    ABSTRACT: Bovine follicle-stimulating hormone (boFSH) is a heterodimeric glycoprotein that belongs to the pituitary gonadotropins. Bioactive FSH is composed of alpha and beta subunits which require extensive N-glycosylation and sialylation. The mammary gland of transgenic livestock is an attractive source for the synthesis of post-translationally modified proteins. Two mammary gland-specific gene constructs with the cDNA for the boFSH alpha (boFSHalpha) and beta (boFSHbeta) subunits controlled by bovine alpha-s1 casein regulatory sequences were co-microinjected into fertilized rabbit oocytes. Two FSHalpha/FSHbeta double transgenic rabbit lines were established. The transgene expression was strictly lactation and mammary gland specific. Protein analysis revealed the presence of the boFSH heterodimer in the milk of transgenic rabbits showing a molecular weight similar to that of purified pituitary gland derived boFSH (boFSH-P). Subunit specific antibodies detected both polypeptides with the expected molecular sizes. Biochemical characterization demonstrated the expected isoelectric points of the recombinant boFSH. The presence of the post-translationally added terminal sialic acid residues was indicated by wheat germ agglutinin (WGA) lectin Western blotting. The biological activity of the recombinant mammary gland produced boFSH was determined using a FSH-dependent reporter cell line. The bioactivity of the recombinant boFSH was comparable to that of purified boFSH-P.
    Full-text · Article · Dec 2002 · Molecular Reproduction and Development
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