title: Kinetic characterization of human thyroperoxidase. Normal and pathological enzyme expression in Baculovirus system: a molecular model of functional expression.
abstract: BACKGROUND
  Human thyroperoxidase (hTPO) is a membrane-bound glycoprotein located at the apical membrane of the thyroid follicular cells which catalyzes iodide oxidation and organification in the thyroglobulin (TG) tyrosine residues, leading to the thyroid hormone synthesis by coupling of iodotyrosine residues. Mutations in hTPO gene are the main cause of iodine organification defects (IOD) in infants.
  
  
  METHODS
  We investigated the functional impact of hTPO gene missense mutations previously identified in our laboratory (p.C808R, p.G387R and p.P499L). In order to obtain the whole wild-type (WT) coding sequence of hTPO, sequential cloning strategy in pGEMT vector was carried out. Then, site-directed mutagenesis was performed. WT and mutant hTPOs were cloned into the pAcGP67B transfer vector and the recombinant proteins were expressed in Baculovirus System, purified and characterized by SDS-PAGE and Western blot. Moreover, we report for the first time the kinetic constants of hTPO, of both WT and mutant enzymes.
  
  
  RESULTS
  The functional evaluation of the recombinant hTPOs showed decreased activity in the three mutants with respect to WT. Regarding to the affinity for the substrate, the mutants showed higher Km values with respect to the WT. Additionally, the three mutants showed lower reaction efficiencies (Vmax/Km) with respect to WT hTPO.
  
  
  CONCLUSIONS
  We optimize the expression and purification of recombinant hTPOs using the Baculovirus System and we report for the first time the kinetic characterization of hTPOs.
authors.affiliation: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: Fiorella S
authors.initials: FS
authors.lastname: Belforte
authors.affiliation: Instituto de Nanobiotecnología (NANOBIOTEC, CONICET-UBA), Cátedra de Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Targovnik
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas "Prof. Alejandro C. Paladini" (IQUIFIB, CONICET-UBA) and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: González-Lebrero
authors.affiliation: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: Carolina
authors.initials: C
authors.lastname: Osorio Larroche
authors.affiliation: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: Cintia E
authors.initials: CE
authors.lastname: Citterio
authors.affiliation: Unidad de Medicina Molecular-Departamento de Medicina, IBMCC and IBSAL, Universidad de Salamanca-CSIC, 37007 Salamanca, España.
authors.forename: Rogelio
authors.initials: R
authors.lastname: González-Sarmiento
authors.affiliation: Instituto de Nanobiotecnología (NANOBIOTEC, CONICET-UBA), Cátedra de Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: María V
authors.initials: MV
authors.lastname: Miranda
authors.affiliation: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
authors.forename: Héctor M
authors.initials: HM
authors.lastname: Targovnik
authors.affiliation: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina. Electronic address: crivolta@ffyb.uba.ar.
authors.forename: Carina M
authors.initials: CM
authors.lastname: Rivolta
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000302:isolation & purification; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000201:enzymology*
chemical_list: D001324:Autoantigens; D033862:Iron-Binding Proteins; D011994:Recombinant Proteins; C056658:TPO protein, human; D007453:Iodide Peroxidase
keywords: Baculovirus System; Expression; Human thyroperoxidase; Iodide organification defects; Kinetic characterization; Thyroperoxidase gene