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Artículo

Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli

Santos, JavierIcon ; Antón, Erica A.; Marino Buslje, Cristina; Valdez, Silvina NoemiIcon ; Villanueva, Ana L.; Sica, Mauricio PabloIcon ; Iacono, Ruben FranciscoIcon ; Maffia, Paulo CesarIcon ; Poskus, EdgardoIcon ; Ermacora, Mario RobertoIcon
Fecha de publicación: 06/2000
Editorial: Portland Press
Revista: Biotechnology and Applied Biochemistry
ISSN: 0885-4513
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Otras Biotecnologías de la Salud

Resumen

Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.
Palabras clave: Gad , Threonin , Recombinant , Thiorredoxin
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
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URI: http://hdl.handle.net/11336/41519
URL: https://iubmb.onlinelibrary.wiley.com/doi/10.1042/BA19990103
DOI: http://dx.doi.org/10.1042/BA19990103
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Articulos(SEDE CENTRAL)
Articulos de SEDE CENTRAL
Citación
Santos, Javier; Antón, Erica A.; Marino Buslje, Cristina; Valdez, Silvina Noemi; Villanueva, Ana L.; et al.; Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli; Portland Press; Biotechnology and Applied Biochemistry; 31; 3; 6-2000; 205-212
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