title: Development of a screening system for inteins active in protein splicing based on intein insertion into the LacZα-peptide.
abstract: Protein 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. Both the cis-variant of the M86 mutant of the Ssp DnaB intein and a split form undergoing protein trans-splicing gave rise to formation of blue colonies in the β-galactosidase read-out. Furthermore, we report the two novel, naturally split VidaL T4Lh-1 and VidaL UvsX-2 inteins whose N-terminal fragments consist of only 15 and 16 amino acids, respectively. Initial biochemical characterization with the LacZα host system of these inteins further underlines its utility. Finally, we used the LacZα host system to rapidly identify amino acid substitutions from a small randomized library at the structurally conserved intein position 2 next to the catalytic center, that are tolerated for protein splicing activity of the M86 intein. These findings demonstrate the potential of the system for initial testing and directed evolution of inteins.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Neugebauer
authors.forename: Jana K
authors.initials: JK
authors.lastname: Böcker
authors.forename: Julian C J
authors.initials: JC
authors.lastname: Matern
authors.forename: Shmuel
authors.initials: S
authors.lastname: Pietrokovski
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
mesh_terms: D000595:Amino Acid Sequence; D015723:Gene Library; D047668:Inteins / Q000235:genetics*; D007763:Lac Operon; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D019154:Protein Splicing; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D010455:Peptides; D001616:beta-Galactosidase