Recent developments in intein-mediated protein splicing and their applications in bioengineering.

Abstract

Inteins are self-excising protein elements that catalyze their own removal from host polypeptides and mediate the ligation of the surrounding exteins, generating mature and functional proteins without the requirement for external cofactors or energy sources. Since their discovery, inteins have attracted considerable interest due to their unique catalytic mechanisms, structural diversity, and broad applicability. Extensive progress in deciphering the molecular basis of cis- and trans-splicing inteins has facilitated the rational design and engineering of improved variants with enhanced efficiency, controllability, and substrate tolerance. These advances have significantly expanded their practical utility. Distinctive features such as compact architecture, high fidelity of splicing, orthogonal activity, minimal cytotoxicity, and irreversible function position inteins as powerful molecular tools in biotechnology and biomedicine. Their applications are increasingly diverse, ranging from fundamental protein engineering tasks such as protein purification, site-specific modification, and selenoprotein production to more translational uses, including microbial drug targeting, intein-based biosensing, targeted gene delivery, and therapeutic gene editing. In addition, conditional inteins, engineered to respond to environmental or molecular cues, are opening new avenues in synthetic biology and biomedicine, enabling precise control over protein function in complex cellular contexts. This review provides a comprehensive overview of intein biology, classification, and mechanistic insights, followed by a discussion of recent developments in their biotechnological and biomedical applications. Particular attention is given to challenges that continue to limit broader adoption, such as incomplete splicing, extein compatibility issues, and context-dependent efficiency. Finally, we highlight emerging opportunities in the field, including the computational design of orthogonal intein libraries, integration with high-throughput screening methods, and the incorporation of artificial intelligence to accelerate intein engineering and application discovery. Collectively, these developments underscore the transformative potential of inteins as versatile molecular tools poised to impact diverse areas of life sciences and therapeutic innovation.