当前位置： SCI文献检索 > ENDOCRINOLOGY期刊下所有文献 > Expression of biologically active human thyrotropin (hTSH) in a baculovirus system: effect of insect cell glycosylation on hTSH activity in vitro and in vivo.

Expression of biologically active human thyrotropin (hTSH) in a baculovirus system: effect of insect cell glycosylation on hTSH activity in vitro and in vivo.

Abstract：

: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 alpha-subunit and a hTSH beta-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.

journal_name

Endocrinology

journal_title

Endocrinology

authors

Grossmann M,Wong R,Teh NG,Tropea JE,East-Palmer J,Weintraub BD,Szkudlinski MW

doi

10.1210/endo.138.1.4897

subject

Has Abstract

pub_date

1997-01-01 00:00:00

pages

92-100

issue

eissn

0013-7227

issn

1945-7170

journal_volume

138

pub_type

杂志文章

在线工具

Expression of biologically active human thyrotropin (hTSH) in a baculovirus system: effect of insect cell glycosylation on hTSH activity in vitro and in vivo.