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A Small‐Molecule–Protein Interaction System with Split‐Ubiquitin as Sensor
Author(s) -
Dirnberger Dietmar,
Unsin Gabriele,
Schlenker Stephan,
Reichel Christoph
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500544
Subject(s) - small molecule , linker , cyclin dependent kinase , ubiquitin , chemistry , protein–protein interaction , fusion protein , chromosomal translocation , ubiquitin conjugating enzyme , biochemistry , computational biology , microbiology and biotechnology , biology , ubiquitin ligase , cell cycle , gene , computer science , recombinant dna , operating system
The identification of receptors for small molecules is of great pharmaceutical importance for drug‐discovery research. Several systems for the identification of protein–small‐molecule interactions have been developed in the past. These were modifications of the classical yeast two‐hybrid system, relying on a transcriptional read‐out following nuclear translocation of the complex. Here we present a novel three‐hybrid technology based on the split‐ubiquitin system for the analysis of protein–small‐molecule interactions independently of a nuclear translocation of the complex. The performance of the system is compared to a method based on the classical yeast two‐hybrid system by using a chemical inducer of dimerization (CID) comprised of methotrexate linked to dexamethasone. Steric issues are addressed by varying the linker length of the compounds, as well as by comparing the orientation of fusion proteins. The system is further extended to the analysis of a small‐molecule inhibitor of human PCTAIRE protein kinase 3, which is related to cyclin‐dependent kinases (CDKs), an important class of pharmaceutical targets.