Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System

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.

Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

“…6) [41]. Thus, Npu DnaE(C+1S) probably requires further improvement for the practical use, for example by directed evolution or rational design [22,23,25].…”

“…6) [41]. Thus, NpuDnaE(C+1S) probably requires further improvement for the practical use, for example by directed evolution or rational design [22,23,25]. Understanding the structure-function relationship of inteins at the splicing junctions might enable us to rationally design inteins with designed features, thereby widening potential applications.…”

“…To overcome the bottleneck imposed by the junction problem of inteins, several groups attempted to engineer more promiscuous inteins by directed evolution or to identify more robust and promiscuous inteins from natural sources [14,[21][22][23][24][25][26]. However, the substrate specificities of various inteins, namely the junction sequence dependencies, have not been thoroughly investigated except for only a very few inteins [8,14,20].…”

Ligation of multiple protein domains using orthogonal inteins with non‐native splice junctions

“…As an approach to bypass this problem, Pinto et al ( 2020 ) successfully built a library of orthogonal inteins with different sequence requirements on the ligation site, where to find suitable candidates whose native splice junction sequences match the desired target extein. An alternative approach has been to identify from natural sources (Cheriyan et al, 2013 ; Ellilä et al, 2011 ) or engineer (Appleby‐Tagoe et al, 2011 ; Aranko et al, 2014 ; Beyer et al, 2020 ; Beyer & Iwaï, 2019 ; Stevens et al, 2017 ) more promiscuous inteins able to efficiently react in non‐natural sequence contexts. Interestingly, the group of Iwaï carried out a systematic investigation to characterize the splicing efficiencies of a panel of inteins when the −1 and +2 positions were replaced by each of the remaining 19 amino acids (Oeemig et al, 2020 ).…”

Evaluating the Potential for Cross-Interactions of Antitoxins in Type II TA Systems

“…Toxin proteins are able to manipulate their bacterial host cells in powerful ways. This drives great interest in understanding their functions and the potential to utilize them for biotechnology and bacterial control strategies [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ].…”