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 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 -splicing split intein and the -splicing mini-intein.
View Article and Find Full Text PDFProtein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N- S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step.
View Article and Find Full Text PDFNonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation domains is only poorly understood.
View Article and Find Full Text PDFProtein splicing by inteins has found diverse applications in biotechnology, protein chemistry and chemical biology. Inteins display a wide range of efficiencies and rates unpredictable from their amino acid sequences. Here, we identified positions T22S and S35 in the LacZα peptide as intein insertion sites that strictly require protein splicing, in contrast to cleavage side-reactions, to allow for complementation of β-galactosidase activity.
View Article and Find Full Text PDFMethods Mol Biol
September 2015
Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment.
View Article and Find Full Text PDFProtein 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.
View Article and Find Full Text PDFCyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split-intein-mediated SICLOPPS technology provides a generic access to fully genetically encoded head-to-tail cyclized peptides and large libraries thereof (SICLOPPS=split-intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery.
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