Protein trans-splicing is a naturally occurring process in which two protein fragments are ligated by a reaction between two intein domains, called split inteins. Despite their usefulness in research, the reactivity and structure of only a few split inteins have been studied. We used cell-based kanamycin selection and next-generation sequencing (NGS) to simultaneously measure the splicing reactivity of 298 N-intein–C-intein combinations derived from the DnaE gene of cyanobacteria. Additionally, we confirmed the splicing activities by measuring the growth of cells individually harboring each split intein under kanamycin selection. Overall, the N-intein–C-intein combinations were promiscuous in their trans-splicing activities, although certain combinations did not splice actively. These results and the NGS-based analysis in this research would be helpful for the development of novel split inteins and further understanding of the trans-splicing mechanism.

蛋白质反式剪接是自然发生的过程，其中两个蛋白质片段通过两个内含肽域之间的反应连接在一起，这称为分裂内含肽。尽管它们在研究中有用，但仅研究了少数分裂内含子的反应性和结构。我们使用基于细胞的卡那霉素选择和下一代测序（NGS）来同时测量源自蓝细菌DnaE基因的298种N-intein-C-intein组合的剪接反应性。此外，我们通过测量在卡那霉素选择下分别携带每个分裂内含子的细胞的生长来确认剪接活性。总体而言，N-内含子-C-内含子的组合在其跨接活动中是混杂的，尽管某些组合没有主动剪接。