Split-ubiquitin based reporter systems and methods of their use

Details Split-ubiquitin based reporter systems and methods of their use Split-ubiquitin based reporter systems and methods of their use

G - Physics

01

N

G01N 33/68 (2006.01) G01N 33/542 (2006.01)

Patent

CA 2417888

Methods and reagents for the detection and selection of two interacting- polypeptides, especially integral membrane proteins and transcription factors, by monitoring the reassembly of ubiquitin amino-terminal and carboxy-terminal chimeric polypeptide fragments are disclosed. Negative selection against an N- end rule-labilized marker released following ubiquiting reassembly allows direct selection of the interacting polypeptide pair. Methods to identify agonists and antagonists for certain protein-protein interactions; methods and reagents/kits for identifying proteins that binds a target protein are also provided. The dynamic and adaptable nature of the assay allows adaptation to a number of applications - such as probing the molecular environment of cellular membrane proteins in vivo.

Cette invention concerne des méthodes et des réactifs permettant de détecter et de sélectionner deux polypeptides d'interaction, en particulier des protéines intrinsèques membranaires et des facteurs de transcription, en surveillant le ré-assemblage de le N-terminal d'ubiquitine et de fragments de polypeptide chimère à terminal carboxy. La sélection négative par rapport à un marqueur labilisé de la règle de la terminaison N, libéré à la suite d'un ré-assemblage d'ubiquitine, permet de sélectionner directement la paire de polypeptides d'interaction. L'invention concerne également des méthodes d'identification d'agonistes et d'antagonistes pour certaines interactions protéine-protéine, ainsi que des méthodes et des réactifs/trousses permettant d'identifier des protéines qui se lient à une protéine cible. De par sa nature dynamique et évolutive, cet essai convient pour de multiples applications, telles que des sondages in vivo dans le milieu moléculaire de protéines intrinsèques.

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