In recent years, split inteins have seen widespread use as molecular platforms for the design of a variety of peptide and protein chemistry technologies, most notably protein ligation. The development of these approaches is dependent on the identification and/or design of split inteins with robust activity, stability, and solubility. Here, we describe two approaches to characterize and compare the activities of newly identified or engineered split inteins. The first assay employs an E. coli-based selection system to rapidly screen the activities of many inteins and can be repurposed for directed evolution. The second assay utilizes reverse-phase high-performance liquid chromatography (RP-HPLC) to provide insights into individual chemical steps in the protein splicing reaction, information that can guide further engineering efforts. These techniques provide useful alternatives to common assays that utilize SDS-PAGE to analyze splicing reaction progress.
Keywords: Bacterial selection; Chemical biology; Kinetic analysis; Protein engineering; Protein trans-splicing; RP-HPLC; Split intein.