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Introducción de manijas reactivas de tiol en proteínas etiquetadas con tirosina a través del acoplamiento oxidativo

Paul Huang¹, Wendy Cao¹, Jennifer L Fetzer¹

¹Department of Chemistry, University of California, Berkeley, California 94720, United States.

Journal of the American Chemical Society

|June 27, 2025

Resumen

Una nueva variante de la enzima tirosinasa, megaTYR, permite una eficiente conjugación de proteínas mediante la conversión de los residuos de tirosina en manijas de tiol utilizando DTT. Esto facilita la creación de conjugados anticuerpo-fármaco y otras terapias bioconjugadas.

Área de la Ciencia:

Biotecnología
Ingeniería de proteínas
Biología Química

Sus antecedentes:

La bioconjugación de proteínas específicas del sitio es crucial para el desarrollo de terapias y materiales avanzados.
La enzima tirosinasa facilita la conjugación proteína-proteína a través de los residuos de tirosina y cisteína.
Los métodos de tirosinasa existentes tienen limitaciones con ciertos sustratos de tiol de moléculas pequeñas.

Objetivo del estudio:

Investigar una nueva variante de tirosinasa (megaTYR) para mejorar la bioconjugación de proteínas.
Explorar el uso del DTT para introducir mangos de tiol en las proteínas.
Demostrar la utilidad de este método para crear conjugados de anticuerpos y fármacos y otros conjugados de proteínas.

Principales métodos:

Se utilizó la variante de Bacillus megaterium tirosinase (megaTYR) por su tolerancia a los sustratos de tiol.
Se utiliza DTT para convertir los residuos de tirosina en manijas de tiol reactivas en las proteínas.
Realizó la conjugación posterior de proteínas tioladas con sondas de maleimida, medicamentos y enzimas.

Principales resultados:

MegaTYR demostró una mayor tolerancia a los sustratos de tiol de molécula pequeña como el DTT.
DTT introdujo con éxito manijas de tiol en proteínas etiquetadas con tirosina, evitando la necesidad de incorporar cisteína genética.
Los conjugados anticuerpo-fármaco y anticuerpo-toxina funcionales fueron sintetizados con éxito utilizando anticuerpos monoclonales tiolados (mAbs).

Conclusiones:

La enzima megaTYR y el DTT proporcionan un método fácil para convertir los residuos de tirosina accesibles en etiquetas de tiol.
Este enfoque amplía la aplicabilidad del acoplamiento oxidativo mediado por la tirosinasa para diversos sustratos proteicos.
El método desarrollado ofrece una plataforma versátil para crear nuevas terapias y materiales bioconjugados.

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