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Off-pathway-sensitive protein splicing screening based on a toxin/antitoxin system.

Research paper by Hannes M HM Beyer, Hideo H Iwai

Indexed on: 12 Apr '19Published on: 10 Apr '19Published in: ChemBioChem



Abstract

Protein splicing domains are frequently used engineering tools finding application for the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, 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. Here, we report a stringent screening method for protein splicing activity in cis and trans, exclusively selecting productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the E. 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 protein splicing e.g. from directed evolution approaches or the natural intein sequence space. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.