Search

Summary 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. [ABSTRACT FROM AUTHOR]

Summary: Natural peptide products often contain N-methylated backbones, and such a modification plays a crucial role in making natural peptides peptidase resistant and membrane permeable. Here, we demonstrate the ribosomal synthesis of N-methyl-peptides by means of genetic code reprogramming. Two key technologies, a ribozyme-based de novo tRNA acylation (flexizyme) system and an E. coli reconstituted cell-free translation (PURE) system, were used in order to reassign arbitrarily chosen codons to N α-methylated amino acids (Meaa). Using this combination, we determined the general structural requirement of “accessible”Meaa and demonstrated their multiple incorporations into the nascent peptide chain according to the assignments made on mRNA, giving linear and cyclic N-methyl-peptides in high purities. This platform technology offers a convenient tool for the construction of N-methyl-peptide libraries, potentially leading to the discovery of therapeutic peptides. [Copyright &y& Elsevier]

Summary: Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of “natural product-like” macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53. [ABSTRACT FROM AUTHOR]