Split Inteins as Versatile Tools for Protein Semisynthesis

Abstract

Divide and conquer: Split inteins link their fused polypeptide sequences with a native peptide bond. This protein trans‐splicing (PTS) reaction can be exploited for manifold applications in protein chemistry and biotechnology.

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

“…Split inteins hold a tremendous potential for a wide range of protein engineering approaches (17,19,30). Recently, novel biotechnological applications for split inteins were described.…”

“…applications, for instance, to generate cyclic peptides (16), to generate protein thioesters (17), to label proteins (18,19), and probably most frequently, to cleave off affinity tags for protein purification (20). Particularly to optimize cleavage efficiency, inteins have been mutagenized to become inducible by pH (21), reducing agents (22), or temperature (23) and to separate cleavage activity from splicing.…”

Use of protein trans‐splicing to produce active and segmentally 2H, 15N labeled mannuronan C5‐epimerase AlgE4

“…Thus segmental isotopic labeling of individual domains in the full-length protein is a very attractive possibility to analyze the structure-function relationship of the individual AlgE4 modules. Figure 7 shows an overlay of NMR spectra of the R-module alone and segmentally labeled AlgE4 (A-[ 2 H, 15 N]-R). It demonstrates clearly that the overall structure of the Rmodule is essentially the same, as most chemical shifts are identical.…”

“…Structural studies of biomolecules by NMR have made tremendous progress mainly due to improved recombinant protein expression and 13 C, 15 of novel NMR experiments. 1,2 It has also expanded the scope of NMR with biological macromolecules such as larger proteins by new labeling technology.…”

“…Inteins are intervening peptide sequences, which excise themselves post-translationally and ligate two flanking N-and C-terminal segments (exteins) via a peptide bond. [15][16][17][18][19][20] In EPL the intein variant is cleaved by thiol reagents resulting in an extein with a C-terminal thioester. Together with the other extein with an N-terminal cysteine, the two fragments then ligate like in NCL.…”