Keren-Kaplan, T. et al. EMBO J. advance online publication (11 November 2011).

Cells use ubiquitylation as an important way to regulate proteins, but degradation and deubiquitylation make it difficult to isolate the modified isoforms for studies. Keren-Kaplan et al. code the entire eukaryotic ubiquitylation apparatus in two plasmids and transport it to Escherichia coli, where ubiquitylated proteins are stable. One plasmid contains affinity-tagged ubiquitin and ubiquitin-activating and ubiquitin-conjugating enzymes, and the other includes an affinity-tagged protein of interest and its cognate ubiquitin ligase. The researchers show that the reconstituted system produces proteins with native, stable ubiquitin modifications, and generate several plasmids with ligase and substrate combinations from different organisms. Affinity pulldown using both tags produces milligrams of highly pure ubiquitylated protein for downstream biochemistry or crystallography studies.