HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jones, F G. Articles by Smith, B R. Search for Related Content PubMed PubMed Citation Articles by Jones, F G. Articles by Smith, B R.

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 biotin-labelled 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, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor -aminolevulinic acid was included in the culture medium.

Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO—TPO autoantibody complexes.

This article has been cited by other articles:

				J. Guo, S. M. McLachlan, S. Hutchison, and B. Rapoport The Greater Glycan Content of Recombinant Human Thyroid Peroxidase of Mammalian Than of Insect Cell Origin Facilitates Purification to Homogeneity of Enzymatically Protein Remaining Soluble at High Concentration Endocrinology, March 1, 1998; 139(3): 999 - 1005. [Abstract] [Full Text] [PDF]

				A. Gardas, A. Lewartowska, B. J. Sutton, Z. Pasieka, A. M. McGregor, and J. P. Banga Human Thyroid Peroxidase (TPO) Isoforms, TPO-1 and TPO-2: Analysis of Protein Expression in Graves' Thyroid Tissue J. Clin. Endocrinol. Metab., November 1, 1997; 82(11): 3752 - 3757. [Abstract] [Full Text] [PDF]

				X. Zhang and P. Arvan Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression J. Biol. Chem., October 6, 2000; 275(41): 31946 - 31953. [Abstract] [Full Text] [PDF]