Protein Splicing Mechanisms and Applications

Abstract

SummaryInteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches. IUBMB Life, 57: 469 -476, 2005

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

“…The potential usefulness of self-splicing in protein engineering fueled detailed studies of the mechanism of action of this multistep process. This understanding led to the development of a number of re-engineered intein constructs that are now commercially available allowing semisynthesis of proteins 22,23 and consequently sitespecific incorporation of unnatural amino acids. One of these techniques, EPL, is the focus of this protocol.…”

Protein Engineering Methods and Applications

“…The proteolytic cleavage and ligation activities of inteins have been understood, which resulted in novel intein applications in protein engineering, enzymology, microarray production, target detection and transgene activation in plants. The conversion of inteins into molecular switches was introduced by intein-mediated protein attachment to solid supports for microarray and western blot studies and by linking nucleic acids to proteins and controlled splicing (Perler, 2005). Recent intein-mediated protein engineering applications like protein purification, ligation, cyclization and selenoprotein production have been discussed in detail lately (Elleuche & Poggeler, 2010).…”

Temperature-Sensitive Mutations Made Easy: Generating Conditional Mutations by Using Temperature-Sensitive Inteins That Function Within Different Temperature Ranges

“…At nonpermissive temperatures, it fails to splice and remains within the host protein, leading to the inactivation of that protein. Intein splicing, also known as protein splicing, is a post-translational process in which the intein is precisely excised from a nascent protein precursor, and the two flanking sequences (N and C exteins) are ligated together through a normal peptide bond (Evans and Xu 2002;Anraku et al 2005;Perler 2005). Thus, no intein footprint is left behind after protein splicing; that is, whatever allele a host protein possesses, be it wild type, dominant negative or constitutively active, the same allele will be generated after splicing.…”