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Obtención y caracterización parcial de la proteína humana mutante α-sinucleína G51D y la producción de su anticuerpo policlonal
Preparation and partial characterization of the mutant human α-synuclein protein G51D and the production of its polyclonal antibody
Obtenção e caracterização parcial da proteína humana mutante α-sinucleína G51D e da produção de seu anticorpo policlonal
DOI:
https://doi.org/10.15446/rev.colomb.quim.v52n2.111120Palabras clave:
α-sinucleína, α-sinucleína G51D, pET30a, variante genética, anticuerpo policlonal, enfermedades neurodegenerativas (es)α-synuclein, α-synuclein G51D, pET30a, genetic variant, polyclonal antibody, neurodegenerative diseases (en)
α-sinucleína, α-sinucleína G51D, pET30a, variante genética, anticorpo policlonal, doenças neurodegenerativas (pt)
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La α-sinucleína (SNCA) es una proteína que participa en la formación de agregados, cuya presencia es distintiva para todas las α-sinucleinopatías y algunas adicciones. Las alteraciones en el gen que codifica la SNCA y en la síntesis de esta proteína han sido relacionadas con procesos de agregación que alteran su conformación y adquieren capacidad autoagregante, lo cual está relacionado con su presencia en las neuronas dopaminérgicas y podría ser un factor fisiopatológico significativo en la progresión de enfermedades. Una de las variantes génicas más comunes de la SNCA es la G51D, la cual podría ser un indicador anatomopatológico para enfermedades neuronales y adicciones de larga duración. En el presente estudio se exponen las técnicas de expresión, purificación y caracterización de la proteína recombinante SNCA G51D, además de ensayos de agregación. Así mismo, se detallan las circunstancias para la obtención del anticuerpo de tipo policlonal dirigido hacia la SNCA G51D. También se realizó la optimización para la clonación de esta variante, utilizando el vector pET30a, el cual nos brindó la mayor solubilidad in silico. La proteína recombinante permitió la obtención de un anticuerpo policlonal anti SNCA G51D, que se caracterizó parcialmente y podría ser una herramienta inmunológica significativa en la confirmación de la existencia de proteínas mutantes en muestras de interés.
α-Synuclein (SNCA) is an essential protein that participates in the formation of aggregates, whose presence is distinctive for all α-synucleinopathies and some addictions. Alterations in the gene that codes for SNCA and in the synthesis of this protein have been related to aggregation processes that alter its conformation and acquire self-aggregating capacity, which is related to its presence in neurons and could be a significant pathophysiological factor in the progression of diseases. One of the most common SNCA gene variants is G51D, which could be an anatomopathological indicator for neuronal diseases and long-term addictions. In the present study, the expression, purification and characterization techniques of the recombinant protein SNCA G51D are presented, as well as aggregation assays. Likewise, the circumstances for obtaining the polyclonal type antibody directed towards SNCA G51D are detailed. Optimization was also carried out for the cloning of this variant, using the pET30a vector, which gave us the greatest in silico solubility. The recombinant protein allowed the obtaining of a polyclonal anti SNCA G51D antibody, which was partially characterized and could be a significant immunological tool in confirming the existence of mutant proteins in samples of interest.
A α-sinucleína (SNCA) é uma proteína que participa da formação de agregados, cuja presença é característica de todas as α-sinucleinopatias e de alguns vícios. Alterações no gene que codifica a SNCA e na síntese desta proteína têm sido relacionadas a processos de agregação que alteram sua conformação e adquirem capacidade de autoagregação, o que está relacionado à sua presença nos neurônios e pode ser um fator fisiopatológico significativo na progressão da doença. Uma das variantes mais comuns do gene SNCA é o G51D, que pode ser um indicador anatomopatológico de doenças neuronais e vícios de longo prazo. No presente estudo são apresentadas técnicas de expressão, purificação e caracterização da proteína recombinante SNCA G51D, bem como ensaios de agregação. Da mesma forma, são detalhadas as circunstâncias para obtenção do anticorpo do tipo policlonal direcionado para SNCA G51D. Também foi realizada otimização para a clonagem desta variante, utilizando o vetor pET30a, que nos proporcionou a maior solubilidade in silico. A proteína recombinante permitiu a obtenção de um anticorpo policlonal anti SNCA G51D, que foi parcialmente caracterizado e poderá ser uma ferramenta imunológica significativa na confirmação da existência de proteínas mutantes em amostras de interesse.
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