Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.
Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.
Zmora
Nilli
N
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202, USA.
Kazeto
Yukinori
Y
Kumar
R Sampath
RS
Schulz
Rüdiger W
RW
Trant
John M
JM
D000818:Animals; D019149:Bioreactors; D004330:Drosophila / Q000378:metabolism; D005260:Female; D037201:Follicle Stimulating Hormone, beta Subunit / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D007059:Ictaluridae / Q000378:metabolism*; D037101:Luteinizing Hormone, beta Subunit / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D008297:Male; D006080:Ovarian Follicle / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013737:Testis / Q000187:drug effects; D014158:Transcription, Genetic
D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D037101:Luteinizing Hormone, beta Subunit; D011994:Recombinant Proteins