title: Traceless protein splicing utilizing evolved split inteins.
abstract: Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cell-based selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Steve W
authors.initials: SW
authors.lastname: Lockless
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004258:DNA Polymerase III / Q000378:metabolism; D019143:Evolution, Molecular; D006367:HeLa Cells; D006801:Humans; D047668:Inteins / Q000235:genetics*; D007613:Kanamycin Resistance; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D050737:Proto-Oncogene Proteins c-crk / Q000737:chemistry; D012641:Selection, Genetic; D020040:Trans-Splicing / Q000235:genetics
chemical_list: D050737:Proto-Oncogene Proteins c-crk; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III