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Protein splicing elements, termed inteins, were first identified in 1990. Since then, post-translational protein splicing has been demonstrated and the selfcatalytic mechanism deciphered. The robust nature of these single turnover enzymes is evidenced by the expanding list of naturally occurring variations in the protein splicing mechanism. Protein splicing must be efficient and neutral, and must not cause detrimental effects to the spliced extein; otherwise, selective pressure would lead to intein loss. Inteins are probably ancient elements, but their original function can only be speculated upon, because invasion by homing endonucleases mobilized them into new locations and converted them into selfish DNA. To date, there is no evidence of regulation of protein splicing in native systems. The sporadic distribution of inteins may relate more to the types of genes found in mobile elements capable of spreading inteins, than to the function of those genes. Inteins tend to be found in conserved host protein motifs, which may be due to conservation of homing endonuclease recognition sites, difficulty in removing inteins from essential regions or the ease of accepting an insertion sequence in a conserved substrate or cofactor binding site designed to interact with the environment. The ability to cleave peptide bonds, to ligate protein fragments and to generate carboxy-terminal alpha-thioesters have made inteins the fastest growing tool for protein engineering and biotechnology.

Intein protein-splicing domains are part of the Hint superfamily. This superfamily includes three other characterized families: Hog-Hint and two types of Bacterial intein-like (BIL) domains. Hint domains share the same structure fold and common sequence features, and have similar biochemical activities. They post-translationally auto-process the proteins in which they are present by protein-splicing, self-cleavage or ligation activities. Yet, each Hint family apparently has its own distinct biological role. We discuss the evolution of the different Hint families, the origin of primordial Hint domains themselves, and their possible activities and biological functions.

This chapter discusses the mechanism of the self-catalyzed process by which inteins promote both their own excision from a host protein and the direct linkage of the flanking host protein segments, the N- and C-exteins, by a peptide bond. The majority of inteins have a nucleophilic amino acid at their N-terminus and asparagine at their C-terminus and are linked to a C-extein with an N-terminal nucleophilic amino acid. These canonical inteins promote protein splicing by a four-step mechanism of sequential acyl rearrangements. Non-canonical inteins, which lack either the N-terminal nucleophile or the C-terminal asparagine, promote protein splicing by a variant of this mechanism or promote protein cleavage rather than splicing. A remarkable feature of the protein splicing process is that it involves multiple steps that are chemically autonomous yet proceed in a highly coordinated manner without side reactions unless perturbed by mutation, unnatural exteins, or non-physiological conditions. The factors that may serve to integrate protein splicing into a system that ordinarily operates efficiently without side reactions are discussed.

Our knowledge of biological systems relies increasingly on the ability of quantifying and imaging intracellular signals and events in living subjects. The development of novel methods and advances in biotechnology have provided many basic tools that allow analyses of the complex biological systems in living cells. Since the discovery of protein splicing in 1990, the elucidation of the splicing mechanism and the identification of key amino acid residues involved in the dissection and ligation of the peptide bonds have facilitated the molecular engineering of inteins for different applications in protein chemistry. These include protein purification, protein ligation and peptide cyclization, construction of split reporter proteins, regulation of protein activity, and introduction of non-natural amino acids. In this chapter, we focus on the construction of split reporter proteins and their applications for detecting protein- protein interactions, identification of organelle-localized proteins, growing safer transgenic plants, and screening antimycobacterial agents.

Intein-based bioseparations have become a widely used technique for the purification of single proteins at the research scale. Aspects of these purification methods suggest that they might eventually be used in more advanced applications, including large-scale protein production and high-throughput proteomic studies. Both of these applications require substantial analysis for further development, particularly concerning the economics and practicality of potential future systems. For scale-up to commercial protein production, an analysis has been made using widely accepted assumptions for process design and a computer model of a hypothetical process. It is clear that the intein process has the potential to be economically competitive, but will be more attractive with the use of pH- and temperature-controlled inteins with lowcost buffer systems. In the case of proteomic applications, prototypical devices have been constructed to demonstrate feasibility. Both of these studies indicate a strong potential for inteins to eventually join mainstream technologies in these areas.