Catalytic Tagging System to Study Macro-Molecular Interactions Using Engineered Ubiquitin Ligase and Ubiquitin-Like Proteins to Facilitate Substrate Identification

摘要

The present invention describes an unbiased catalytic tagging system to search for target substrates. Identification of substrates for specific baits is accomplished by utilizing an orthogonal system consisting of an E1 activating enzyme, an E2 ubiquitin-like conjugating enzyme, and baits that are fused to the E2 ubiquitin-like conjugating enzyme. The present invention thus reveals important mutually antagonistic substrates of specific baits.

主权项

1. An assay for the identification of a substrate comprising:
fusing a bait to an E2 ubiquitin-like conjugating enzyme wherein under wild-type conditions said E2 ubiquitin-like conjugating enzyme conjugates a ubiquitin-like protein other than ubiquitin; expressing in a cell said bait fused to said E2 ubiquitin-like conjugating enzyme wherein the cell comprises one or more ubiquitin-like proteins; and identifying a substrate, wherein said substrate binds ubiquitin under wild-type conditions and is identified by selecting said substrate bound to said one or more ubiquitin-like proteins.