title: High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line.
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, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor delta-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.
authors.affiliation: FIRS Laboratories, RSR Limited, Parc Ty Glas, Cardiff, UK.
authors.forename: F
authors.initials: F
authors.lastname: Grennan Jones
authors.forename: A
authors.initials: A
authors.lastname: Wolstenholme
authors.forename: S
authors.initials: S
authors.lastname: Fowler
authors.forename: S
authors.initials: S
authors.lastname: Smith
authors.forename: K
authors.initials: K
authors.lastname: Ziemnicka
authors.forename: J
authors.initials: J
authors.lastname: Bradbury
authors.forename: J
authors.initials: J
authors.lastname: Furmaniak
authors.forename: B
authors.initials: B
authors.lastname: Rees Smith
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007313:Insecta; D007453:Iodide Peroxidase / Q000032:analysis / Q000096:biosynthesis*; D007700:Kinetics; D037102:Lectins; D017924:Nucleopolyhedroviruses; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D018411:Spodoptera; D013961:Thyroid Gland / Q000201:enzymology; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D037102:Lectins; D011994:Recombinant Proteins; D007453:Iodide Peroxidase