A promiscuous split intein with expanded protein engineering applications

Significance Naturally split inteins are important tools in chemical biology and protein engineering, as they provide a rapid and bioorthogonal means to link two polypeptides, termed exteins, together in a near-traceless manner. However, their use is currently limited by sequence constraints imposed by these extein residues. The engineered split inteins reported in this work, Npu GEP and Cfa GEP , demonstrate a marked enhancement in extein tolerance, offering greater promiscuity for splicing nonnative sequences. As such, they are shown to improve two important applications of naturally split inteins: protein cyclization and the chemical tailoring of native chromatin. We expect these promiscuous inteins to find broad use in other applications of split inteins that involve the construction of proteins with well-defined sequences.