Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

Inteins catalyze self‐excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the −1 and +2 positions, often strongly influence the protein‐splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed “QuickDrop”‐cassette mutagenesis for investigating the influences of 20 amino acid types at the −1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure–function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.