Expression and characterization of biologically active ovine FSH from mammalian cell lines.
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.
Mountford
P S
PS
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Brandon
M R
MR
Adams
T E
TE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis* / Q000737:chemistry; D007525:Isoelectric Focusing; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012708:Sertoli Cells / Q000378:metabolism; D012756:Sheep; D013552:Swine; D014162:Transfection
D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone