Abstract
Protein‐splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein‐splicing activity are associated with bottlenecks such as the selection of pseudo‐positive clones arising from off‐pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein‐splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis‐ and trans‐splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed‐evolution approaches or the natural intein sequence space.
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