Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution. A small-size engineered enzyme, ultraID, is presented for proximity-dependent biotinylation, that shows efficient labeling in mammalian cell culture, E. coli and S. cerevisiae.

Automated summary

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has been established as a key technology for studying protein-protein interactions. To improve the efficiency and speed of PDB, various engineered enzymes have been developed, with ultraID being the smallest and most efficient enzyme to date. It allows for high-resolution investigation of protein interaction networks in different types of cells.