Abstract
Intein-mediated protein splicing has become an essential tool in modern biotechnology. Fundamental progress in the structure and catalytic strategies of cis- and trans-splicing inteins has led to the development of modified inteins that promote efficient protein purification, ligation, modification and cyclization. Recent work has extended these in vitro applications to the cell or to whole organisms. We review recent advances in intein-mediated protein expression and modification, post-translational processing and labeling, protein regulation by conditional protein splicing, biosensors, and expression of trans-genes.
Highlights
Protein splicing is a post-translational process by which an intervening polypeptide, called an intein, catalyzes its own excision from the flanking polypeptides, or exteins, as well as ligation of the exteins (Figure 1A).Many inteins are interrupted by homing endonuclease domains similar to those found in mobile introns
We aim to describe in detail the most recent advances in this area, including protein expression and modification, post-translational processing and labeling, protein regulation by conditional protein splicing, biosensors, and the expression of trans-genes
An E. coli-based biosensor was developed to monitor binding between calmodulin and its target peptide M13, using green fluorescent protein (GFP) reconstitution as the reporter, mediated by the artificially split Saccharomyces cerevisiae (Sce) VMAI intein [99]
Summary
Protein splicing is a post-translational process by which an intervening polypeptide, called an intein, catalyzes its own excision from the flanking polypeptides, or exteins, as well as ligation of the exteins (Figure 1A). Callahan and coworkers designed a redox trap into the fused, cis-splicing version of the Ssp DnaE intein, by introducing a Cys (Cys-3) residue in the N-extein This intein could facilitate N-terminal cleavage only under reducing conditions in E. coli and allows for purification of uncleaved precursor and subsequent in vivo cleavage after addition of reducing agents [95]. Umezawa and coworkers applied this sensor design to demonstrate protein-protein interactions in various in vivo systems ranging from E. coli to transgenic animals In their original work, an E. coli-based biosensor was developed to monitor binding between calmodulin and its target peptide M13, using GFP reconstitution as the reporter, mediated by the artificially split Sce VMAI intein [99]. The Ssp DnaE intein was shown to help improve functional Cre fragment complementation [129]
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