Split Inteins: Nature’s Protein Ligases

Abstract

Split inteins carry out a naturally occurring process known as protein trans-splicing, where two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. In the past thirteen years since their discovery, chemists and biologists have utilized split inteins in exogenous contexts for a number of biotechnological applications centered around the formation of native peptide bonds. While many protein trans-splicing technologies have emerged and flourished in recent years, several factors still limit their wide-spread practical use. Here, we discuss the development, applications, and limitations of split intein-based technologies and propose that further advancement in this field will require a more fundamental understanding of split intein structure and function.

Recent progress in intein research: from mechanism to directed evolution and applications

“…Well-established applications of the latter kind are the preparation of protein thioesters as reagents for the expressed protein ligation (EPL) approach [90,91] and the self-cleavage for tag-free affinity purification of proteins [92]. Split inteins are especially attractive tools to join foreign polypeptide sequences prepared by recombinant protein expression and/ or chemical synthesis and have received much attention recently [13,18]. In this section, we highlight a few recent examples of how progress in the biochemical characterization and mechanistic investigation of inteins could be turned into new developments of intein-based applications and their far-reaching potential for research areas such as protein engineering, chemical biology, and cell biology.…”

“…In this section, we highlight a few recent examples of how progress in the biochemical characterization and mechanistic investigation of inteins could be turned into new developments of intein-based applications and their far-reaching potential for research areas such as protein engineering, chemical biology, and cell biology. For a more detailed discussion on these topics, the reader is referred to the more specialized review articles [13,15,[17][18][19][20]93].…”

“…Split inteins can be regarded as nature's protein ligases [13,18]. A protein or a short peptide tag that may include non-proteinogenic chemical groups can be site-specifically installed either at the N-terminus or at the C-terminus of recombinant proteins by means of protein trans-splicing (Fig.…”

“…A number of reviews have focused on recent advances of intein-based applications [13][14][15][16][17][18], and the reader is referred to these articles for a more in-depth study of the subject, as this review will highlight mostly those applications that were stimulated by biochemical and mechanistic insights into protein splicing. Another emerging field is the use of inteins for pharmaceutical purposes [19,20], which is also beyond the scope of this review.…”

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

“…Subtiligase can direct amide bond formation but substrates require an ester-activated C terminus, and its great advantage for proteomics is that almost any N-terminal sequence is reactive (32,33). Native chemical ligation also allows reaction by using a strategically placed cysteine residue, but, like split inteins and sortase, is limited to the N-and C-termini for fusion of the proteins (34)(35)(36), disallowing nonlinear protein architectures. Therefore, SpyLigase, with no cysteine-bearing components and the ability to direct isopeptide bond formation to an internal peptide tag, possesses unique characteristics for protein modification and assembly, although it will be valuable in the future to evolve SpyLigase and its peptide partners for greater speed and yield.…”