Caracterización tridimensional de proteínas de soya mediante electroforesis de dos dimensiones y partición en fases acuosas
Tridimensional soy protein characterization using two–dimensional electrophoresis and partition in aqueous phases
O. Aguilar1, M. Rito–Palomares1* y C. E. Glatz2
1 Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey. Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, México. * Autor para la correspondencia. E–mail: mrito@itesm.mx
2 Department of Chemical and Biological Engineering, 2114 Sweeney Hall, Iowa State University, Ames, Iowa 50011–2230, U.S.A.
]]>Recibido 14 de Noviembre 2008
Aceptado 12 de Diciembre 2008
Resumen
El conocimiento general de las propiedades moleculares de las potenciales proteínas contaminantes favorece la selección y el diseño de estrategias adecuadas para la recuperación de proteínas recombinantes. Una nueva estrategia experimental resultado de la combinación de electroforesis de dos dimensiones (2DE) cuantitativa con sistemas de dos fases acuosas (SDFA) hidrofóbicos fue empleada para la caracterización tridimensional de proteínas de soya. Los gráficos tridimensionales formados por peso molecular (MR), punto isoeléctrico (pI) e hidrofobicidad superficial (Log Kp) fueron obtenidos usando dos diferentes composiciones de SDFA (poli–etilenoglicol (PEG) 3350 (15.7%)–sulfato de sodio (8.9%)–NaCl (3%) y PEG 3350 (14.8%)–fosfato de potasio (10.3%)–NaCl (3%)). Los dos sistemas evaluados resultaron en diferentes valores de hidrofobicidad para el mismo extracto protéico, sugiriendo una alta influencia de la sal empleada para formar los SDFA en el comportamiento de partición de las proteínas. La presencia de proteínas dominantes derivadas de las dos principales proteínas de almacenamiento, limitó el número de proteínas detectadas en los geles, y por tanto el número de puntos caracterizados en 3D. La identificación de las principales proteínas contaminantes y su proporción relativa en las graficas tridimensionales, representa una etapa inicial para la selección de mejores estrategias de purificación o incluso la selección de un hospedero más adecuado en el desarrollo de procesos biotecnológicos.
Palabras clave: electroforesis 2D, sistemas de dos fases acuosas, soya, proteína.
Abstract
General knowledge of the molecular properties of the potential contaminant proteins benefits the selection and design of suitable strategies for the recovery of recombinant proteins. A novel experimental approach that resulted from the combination of quantitative 2D electrophoresis (2–DE) with hydrophobic partitioning in aqueous two–phase systems (ATPS) was applied for the three–dimensional characterization of soybean proteins. The three dimensional scatter plots of molecular weight (MR), isoelectric point (pI) and surface hydrophobicity (log KP) were obtained using two different ATPS compositions, PEG 3350 (15.7%)–sodium sulfate (8.9%)–NaCl (3%) and PEG 3350 (14.8%)–potassium phosphates (10.3%)–NaCl (3%). Molecular properties of soybean proteins were obtained (MR, pI and log KP) simultaneously using two different ATPS resulting in two different protein profiles, suggesting a high influence of the phase–forming salt on the partitioning behavior of soybean proteins. The presence of dominant proteins derived from two main storage proteins limited the number of spots detected in gels and consequently the number of 3D spots characterized. The identification of the major contaminants proteins and their relative concentration depicted in the tridimensional graph represents the first stage in the selection of better strategies for the purification of products or the selection of a potential host in the development of bioprocesses.
]]> Keywords: 2D–electrophoresis; aqueous two–phase systems; soybean; protein characterization.
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Agradecimientos
Los autores agradecen el apoyo financiero a la Cátedra de Investigación de Bioingenieria y Nanobioparticulas (CAT161) del Tecnológico de Monterrey y al Departamento de Ingeniería Química y Biológica de Iowa State University.
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