High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system.
Superovulation treatment of cows can benefit from the application of very pure recombinant bovine FSH (rbFSH), which is produced in nonmammalian cells. rbFSH is completely free of LH, and therefore can possibly reduce the variability in the results of superovulation. Furthermore, it does not contain brain-tissue-derived proteins and, when produced under serum-free conditions, it is free of other mammalian substances or potentially infectious material. We have produced rbFSH in insect cells, with the ultimate aim of inducing superovulation in cattle. Sf21 insect cells were coinfected with two recombinant baculoviruses, containing the cDNAs of bovine FSH alpha- and beta-subunits respectively. High levels of production of bioactive rbFSH were obtained after cloning cDNA that contained a major part of the 3' untranslated region of the bFSH beta gene. Maximum production of rbFSH 1-5 micrograms/ml (as measured by immunoassay) was obtained 70-90 h after infection. The recombinant material was highly potent in two in vitro bioassays, giving biological activities of 13 IU/ml (Y1 cell rounding assay), 22 IU/ml (Y1 cell cAMP assay), and 23 IU/ml (bovine oocyte maturation inhibition assay), and had a lower but significant activity of 6 IU/ml in the rat Sertoli cell assay. rbFSH was purified by immunoaffinity chromatography, using a monoclonal antibody directed against the human FSH beta-subunit. The purified heterodimer appeared to be homogeneous by SDS-PAGE, whereas the free beta-subunit appeared as a doublet, possibly indicating differently glycosylated forms. Intact heterodimer and both subunits were further identified by western blot analysis, and showed apparent molecular masses of 20 kDa (alpha-subunit), 23 kDa (beta-subunit) and 32.5 kDa (heterodimer). This insect-cell-produced rbFSH did not bind to wheat germ agglutinin, thus indicating that glycosidic side-chains may not contain terminal sialic acid. The relevance of a large 3' untranslated region in bFSH beta cDNA to the level of production of rbFSH, and the possible implications of the pattern of glycosylation for the biological activity of the recombinant hormone are discussed.
van de Wiel
D F
DF
Institute for Animal Science and Health (ID-DLO), Department of Molecular Recognition, Lelystad, The Netherlands.
van Rijn
P A
PA
Meloen
R H
RH
Moormann
R J
RJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism; D018411:Spodoptera
D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone