Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells

Ji Lao Fan, Sai A. Patibandla, Shioko Kimura, Thotakura N. Rao, Rajesh K. Desai, Gattadahalli S. Seetharamaiah, Alexander Kurosky, Bellur S. Prabhakar

    Research output: Contribution to journalArticle

    16 Citations (Scopus)

    Abstract

    Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 μg/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.

    Original languageEnglish (US)
    Pages (from-to)529-536
    Number of pages8
    JournalJournal of Autoimmunity
    Volume9
    Issue number4
    DOIs
    StatePublished - Aug 1996

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    Iodide Peroxidase
    Insects
    Thyroid Gland
    Carbohydrates
    Insect Vectors
    Sf9 Cells
    Galactosamine
    Hashimoto Disease
    Hemin
    Baculoviridae
    Thyroid Diseases
    Serum-Free Culture Media
    Protein Sequence Analysis
    Autoantigens
    Mannose
    Serum
    Oligosaccharides
    Thyroid Hormones
    Human Activities
    Buffers

    Keywords

    • Autoimmunity
    • Expression
    • HPLC
    • Purification
    • TPO

    ASJC Scopus subject areas

    • Immunology
    • Immunology and Allergy

    Cite this

    Fan, J. L., Patibandla, S. A., Kimura, S., Rao, T. N., Desai, R. K., Seetharamaiah, G. S., ... Prabhakar, B. S. (1996). Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells. Journal of Autoimmunity, 9(4), 529-536. https://doi.org/10.1006/jaut.1996.0071

    Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells. / Fan, Ji Lao; Patibandla, Sai A.; Kimura, Shioko; Rao, Thotakura N.; Desai, Rajesh K.; Seetharamaiah, Gattadahalli S.; Kurosky, Alexander; Prabhakar, Bellur S.

    In: Journal of Autoimmunity, Vol. 9, No. 4, 08.1996, p. 529-536.

    Research output: Contribution to journalArticle

    Fan, JL, Patibandla, SA, Kimura, S, Rao, TN, Desai, RK, Seetharamaiah, GS, Kurosky, A & Prabhakar, BS 1996, 'Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells', Journal of Autoimmunity, vol. 9, no. 4, pp. 529-536. https://doi.org/10.1006/jaut.1996.0071
    Fan, Ji Lao ; Patibandla, Sai A. ; Kimura, Shioko ; Rao, Thotakura N. ; Desai, Rajesh K. ; Seetharamaiah, Gattadahalli S. ; Kurosky, Alexander ; Prabhakar, Bellur S. / Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells. In: Journal of Autoimmunity. 1996 ; Vol. 9, No. 4. pp. 529-536.
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