Inteins are auto-processing domains found in organisms from all domains of life. These proteins carry out a process known as protein splicing, which is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds. While the endogenous substrates of protein splicing are specific essential proteins found in intein-containing host organisms, inteins are also functional in exogenous contexts and can be used to chemically manipulate virtually any polypeptide backbone. Given this, protein chemists have exploited various facets of intein reactivity to modify proteins in myriad ways for both basic biological research as well as potential therapeutic applications. Here, we review the intein field, first focusing on the biological context and phylogenetic diversity of inteins, followed by a description of intein structure and biochemical function. Finally, we discuss prevalent intein-based technologies, focusing on their applications in chemical biology, followed by persistent caveats of intein chemistry and approaches to alleviate these shortcomings. The findings summarized herein describe two and a half decades of research, leading from a biochemical curiosity to the development of powerful protein engineering tools.

Inteins: nature's gift to protein chemists

Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag.

Structure and function of legumain in health and disease.

This review article highlights the strategies to successfully perform an efficient solid-phase synthesis of complex peptides including posttranslational modifications, fluorescent labels, and reporters or linking groups of exceptional value for biological studies of several important diseases. The solid-phase approach is the best alternative to synthesize these peptides rapidly and in high amounts. The key aspects that need to be considered when performing a peptide synthesis in solid phase of these molecules are discussed.

/pdf/solid-phase-peptide-synthesis-an-overview-focused-on-the-1nmlt9ppw6.pdf

Solid-phase peptide synthesis: an overview focused on the preparation of biologically relevant peptides

Protein semisynthesis-defined herein as the assembly of a protein from a combination of synthetic and recombinant fragments-is a burgeoning field of chemical biology that has impacted many areas in the life sciences. In this review, we provide a comprehensive survey of this area. We begin by discussing the various chemical and enzymatic methods now available for the manufacture of custom proteins containing noncoded elements. This section begins with a discussion of methods that are more chemical in origin and ends with those that employ biocatalysts. We also illustrate the commonalities that exist between these seemingly disparate methods and show how this is allowing for the development of integrated chemoenzymatic methods. This methodology discussion provides the technical foundation for the second part of the review where we cover the great many biological problems that have now been addressed using these tools. Finally, we end the piece with a short discussion on the frontiers of the field and the opportunities available for the future.

Chemoenzymatic Semisynthesis of Proteins.

Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (-1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(-1) residue, including d-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N-S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (-1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(-1)Phe and Gly(-1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein-intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein-intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.

/pdf/a-functional-interplay-between-intein-and-extein-sequences-27th84go4b.pdf

A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine.

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

Ligation of synthetic peptides to proteins using semisynthetic protein trans-splicing.

Methods in Protein Biochemistry

The present invention relates to an isolated polypeptide comprising at least one intein or at least one intein fragment of said intein, wherein said intein is a naturally split intein with a N-terminal intein fragment split after 14-60 amino acids from the intein's N-terminal end and methods of using the same.

An atypical naturally split intein engineered for highly efficient protein modification

Protein trans-splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL-TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N-terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans-splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50-fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi-synthesis and other intein-related biotechnological applications.

An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling

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