CONTRATADOS
TARGOVNIK Hector Manuel
artículos
Título:
Kinetic characterization of human thyroperoxidase. Normal and pathological enzyme expression in Baculovirus System: A molecular model of functional expression
Autor/es:
BELFORTE, FIORELA SABINA; TARGOVNIK, ALEXANDRA MARISA; GONZÁLEZ-LEBRERO, RODOLFO M.; OSORIO LARROCHE, CAROLINA ; CITTERIO, CINTIA E.; GONZÁLEZ-SARMIENTO, ROGELIO; MIRANDA, MARÍA V.; TARGOVNIK, HÉCTOR M.; RIVOLTA CARINA M.
Revista:
MOLECULAR AND CELLULAR ENDOCRINOLOGY.
Editorial:
ELSEVIER IRELAND LTD
Referencias:
Lugar: Amsterdam; Año: 2015 vol. 404 p. 9 - 9
ISSN:
0303-7207
Resumen:
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.
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