Self-Excising Proteins: Dual-Intein, Intein-2A, and Intein-Ubiquitin for Coordinated Multi-Gene Expression in Synthetic Biology

The production of multiple proteins using a single open reading frame (sORF)/polyprotein system is a powerful strategy for coordinated multi-protein expression in eukaryotes. The most widely used approach relies on 2A peptides, but conventional 2A systems suffer from several limitations, and their viral origin makes them less than ideal for commercial crop biotechnology applications. Self-excising protein (SEP) modules are a promising alternative that enables coordinated production of multiple proteins from a single ORF encoding a polyprotein precursor. SEPs provide distinct advantages over conventional systems and effectively address many of the limitations inherent to the 2A approach. An SEP module is a fusion protein composed of an N-terminal excising domain (NED) and a C-terminal excising domain (CED) joined by a peptide linker. Using this architecture, a panel of SEP modules has been developed by pairing an engineered intein (serving as the NED) with various CEDs, including a second engineered intein, a 2A-like peptide, and ubiquitin. These modules release multiple proteins from the polyprotein precursor with nearly stoichiometric expression and clean cleavage. Coordinated coexpression using SEPs has been successfully demonstrated in several eukaryotic systems, including yeast, mammalian cells, and plants. This review offers a comprehensive analysis of the SEP technology while underscoring its major applications.