PMID: 8938592Oct 1, 1996Paper

High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line

Journal of Molecular Endocrinology
F Grennan JonesB Rees Smith

Abstract

Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotinlabelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r = 0.96, P < 0.001, n = 50 and r = 0.99, P < 0.001, n = 80 respectively). In addition, rTP...Continue Reading

Citations

Aug 25, 2000·Thyroid : Official Journal of the American Thyroid Association·P GutB Rees Smith
Oct 31, 2003·Bioscience, Biotechnology, and Biochemistry·Tetsuya TanakaKei-ichi Shimazaki
May 18, 2000·Biochemical and Biophysical Research Communications·K ShinB Lönnerdal
Jan 19, 2005·Clinical and Experimental Immunology·G D ChazenbalkS M McLachlan
Oct 5, 2013·Endocrine Reviews·Sandra M McLachlan, Basil Rapoport
Dec 29, 2000·International Reviews of Immunology·S M McLachlan, B Rapoport
Aug 5, 2000·The Journal of Biological Chemistry·X Zhang, P Arvan
Oct 16, 1999·Autoimmunity·F Grennan JonesB Rees Smith
Aug 16, 2005·Archives of Biochemistry and Biophysics·Jean Ruf, Pierre Carayon

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