Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae.

摘要: In budding yeast and higher eukaryotic genomes, there are, respectively, 50 up to 400 or more distinct genes that encode for ubiquitin‐ligases, ∼15–90 ubiquitin isopeptidases (TM RJD, Semple et al., 2003). This puts ubiquitylation on par with phosphorylation as the most common reversible posttranslational modifications in cells. A key challenge has met limited success date is identify proteins are substrates this large collection of enzymes. To begin address daunting challenge, we sought ubiquitylated potential 26S proteasome. Here, describe a two‐step affinity purification protocol uses strain expresses hexahistidine‐tagged ubiquitin. first step, native cell lysate was chromatographed UBA domain‐containing matrix binds preferentially K48‐linked multiubiquitin chains. Free presumably monoubiquitylated did not bind column, whereas proteasome were enriched. second domain–binding subjected immobilized metal ion chromatography (IMAC) under denaturing conditions magnetic nickel beads, resulting >3000‐fold enrichment conjugates relative crude extract.