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Expression of Biologically Active Human Thyrotropin (hTSH) in a Baculovirus System: Effect of Insect Cell Glycosylation on hTSH Activity in Vitro and in Vivo¹

From the Department of Medicine, Division of Endocrinology, University of Maryland Medical School and the Institute of Human Virology, Medical Biotechnology Center, Baltimore, Maryland 21201; and the Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases (R.W., N.G.T., J.E.T., J.E.-P.), NIH, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: M. W. Szkudlinski, M.D., Ph.D., Laboratory of Molecular Endocrinology, Institute of Human Virology, Medical Biotechnology Center, 725 West Lombard Street, N457, Baltimore, Maryland 21201. E-mail: szkudlin{at}umbi.umd.edu

To obtain large amounts of hTSH and to study the role of the N-linked oligosaccharides for its biological activity, hTSH was produced using recombinant baculovirus containing the human -subunit and a hTSH ß-minigene, respectively, both under the control of the polyhedrin promoter. Expression in insect cells was 800-1000 ng/ml, 30-fold higher than in our optimized mammalian transient transfection system using Chinese hamster ovary (CHO) cells (20–50 ng/ml). The in vitro activity of insect-cell expressed hTSH (IC-hTSH) was increased 5-fold compared with CHO-hTSH, judged by the ability to induce cAMP production in CHO cells stably transfected with the hTSH receptor (JP09) and the rat thyroid cell line FRTL-5, as well as growth promotion in FRTL-5 cells. Lectin binding and enzymatic desialylation studies suggested that in contrast to CHO-hTSH, IC-hTSH lacked complex-type oligosaccharides terminating with sialic acid but contained predominantly high mannose-type oligosaccharides. The in vitro activity of CHO-hTSH also increased 5- to 6-fold upon treatment of the hTSH-producing cells with the oligosaccharide processing inhibitors swainsonine and castanospermine, which inhibit formation of complex, terminally sialylated oligosaccharides, and upon enzymatic desialylation. In contrast, insect cell-expression or treatment with processing inhibitors did not affect TSH receptor binding. Despite the higher in vitro activity, IC-hTSH had a much lower in vivo activity than CHO-hTSH, due to rapid clearance from the circulation. In summary, this study shows for the first time that relatively high levels of recombinant hTSH with high in vitro bioactivity can be produced in a baculovirus system. Cell-dependent glycosylation is a major factor that determines the final in vivo biopotency of recombinant glycoproteins, a finding that should be of general relevance for all insect cell-produced glycosylated proteins. Although not suitable for clinical use, highly bioactive recombinant hTSH derived from high expression in insect cells should be useful in defining structure-function relations of hormone analogs.

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