Incorporación química de la sonda fluorescente FlAsH en la proteína Hha: aplicación al estudio del complejo Hha/H-NS
Incorporación química de la sonda fluorescente FlAsH en la proteína Hha: aplicación al estudio del complejo Hha/H-NS
Incorporación química de la sonda fluorescente FlAsH en la proteína Hha: aplicación al estudio del complejo Hha/H-NS
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Cordeiro, Tiago
Granados Acevedo, Catalina
Pons, Miquel
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Universidad Colegio Mayor de CundinamarcaCitación
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El objetivo de esta investigación fue realizar estudios de incorporación de la sonda FlAsH como posible herramienta para el estudio in vitro e in vivo de la proteína Hha y de su interacción con H-NS. Se construyó una proteína con la secuencia específica “CCPGCC” capaz de unir la sonda fluorescente FlAsH; posteriormente se desarrollaron procesos de transformación, expresión y purificación, con los cuales se evidenció que a pesar de introducir una secuencia adicional no nativa a la proteína, se pudo obtener proteína en buena cantidad, estabilidad, rendimiento y con un alto nivel de pureza. La optimización del protocolo de incorporación del FlAsH se hizo teniendo en cuenta el rendimiento y el tiempo de reacción. El complejo FlAsH/HhaCCPGCC se caracterizó mediante fluorescencia y se comprobó mediante MALDI TOF. La incorporación HhaCCPGCC1/ FlAsH no se obtuvo en un alto porcentaje como se esperaba, por esta razón no se pudieron hacer los estudios de interacción entre el complejo de proteínas Hha y H-NS. Chemical Incorporation of Fluorescent Probe FlAsH on the Hha Protein: Application to the Study of Complex Hha/H-NS The objective of this investigation was the development of studies of incorporation of the probe F1AsH as a possible tool for the study in vitro and in vivo of the protein Hha and the interaction with H-NS. A protein was constructed with the specific sequence “CCPGCC” able to join the fluorescent molecular probe FLAsH; later, processes of transformation, expression, and purification were developed, with which it was demonstrated that even with the introduction of an additional no native sequence in the protein, it could be obtain protein in a good amount, stability, yield and whit a high level of purity. The optimization of the protocol of incorporation of FLAsH was made considering the yield and the time of reaction. FLAsH was characterized with fluorescence and was verified with MALDI TOF. The incorporation HhaCCPGCC1/ FLAsH could not be obtained in a high percent as it was expected; therefore, the studies of interaction between the protein complex Hha and H-NS could not be done.
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