The proteasome-related Connectase is a fast and specific protein ligase

Genetic methods allow the recombinant production of any protein of interest, but yield the full-length construct in one step and are limited to native amino acids. For the "on demand" generation of chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins, these proteins must be modified using chemical1,2, (split) intein2,3, split domain4 or enzymatic methods5. While each of these options comes with its own advantages and drawbacks, ligase enzymes are often used where small ligation motifs and good chemoselectivity are required. However, applications with the reference enzyme Sortase A are impeded by poor catalytic efficiencies, low substrate specificities and side reactions6,7. Here, we present the discovery of Connectase, a monomeric proteasome homolog that ligates substrates via a highly conserved KDPGA motif originally identified in methyltransferase A (MtrA), a key enzyme in archaeal methanogenesis. Connectase displays nanomolar affinity and thus great specificity for its substrates, allowing efficient protein-protein ligations even in complex solutions and at low substrate concentrations. Compared to an optimized Sortase variant, Connectase catalyzes such ligations at substantially higher rates, with higher yields but without detectable side reactions and thus presents a valuable new tool for protein conjugations.