Expression of the genes encoding bovine LH in a line of Chinese hamster ovary cells

Abstract

Summary. Synthesis of biologically active LH is complex, due in part to its hetero-dimeric subunit structure and to the numerous post-translation modifications of each subunit. Through the use of mammalian expression vectors we have been able to introduce the bovine α subunit and LH-β genes into a Chinese hamster ovary cell line deficient in dihydrofolate reductase. The bovine genes are actively expressed and the Chinese hamster ovary cells secrete biologically active LH. The expression vector containing the bovine α subunit gene also contains a modified mouse gene encoding dihydrofolate reductase, permitting the use of methotrexate to amplify selectively the bovine α subunit gene after its integration into the genome of the Chinese hamster cells. This provides a novel means for assessing the importance of α subunit concentration with respect to assembly of the heterodimer. In addition, methotrexate selection leads to the over-production of LH (10 µg/106 cells/24 h). Finally, because the bovine LH produced in the Chinese hamster ovary cells is glycosylated, this transfection system can be used in conjunction with in-vitro mutagenesis to determine whether site-specific changes in glycosylation have an effect on subunit assembly and biological activity. This transfection approach therefore offers multiple avenues to explore further the molecular mechanisms underlying the complex biosynthetic pathway of bovine LH.