title: DIVERSE System: De Novo Creation of Peptide Tags for Non-enzymatic Covalent Labeling by In Vitro Evolution for Protein Imaging Inside Living Cells.
abstract: Polypeptide-tag/small-molecule pairs for specific cellular protein labeling are useful for visualizing cellular proteins and controlling their activity. Here, we report the development of an in vitro evolution-based (poly)peptide tag identification system named the DIVERSE (Directed In Vitro Evolution of Reactive peptide tags via Sequential Enrichment) system. In this system, an extremely diverse (10(14)) library of peptide tags, displayed by covalent attachment to their encoding cDNAs, is continuously prepared from the DNA library in a one-pot approach. Using this system, we demonstrated de novo creation of non-enzymatically covalent-labeling peptide tags for a synthetic small-molecule target from a random peptide library. Protein labeling with these tags was applicable to N- and C-terminal fusions, multiple different proteins and fluorophores, and intracellular labeling. The DIVERSE system can be used not only for the de novo creation of polypeptide tags but also sequence optimization of existing polypeptide tags from extremely diverse libraries.
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan. Electronic address: t-kawakami@aist.go.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawakami
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Ogawa
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Natsume
mesh_terms: D002477:Cells / Q000378:metabolism; D003584:Cytological Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D019151:Peptide Library; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D019151:Peptide Library; D011506:Proteins