In Vitro Selection of Functional Lantipeptides

Abstract

In this report we present a method to identify functional artificial lantipeptides. In vitro translation coupled with an enzyme-free protocol for posttranslational modification allows preparation of more than 1011 different lanthionine containing peptides. This diversity can be searched for functional molecules using mRNA-lantipeptide display. We validated this approach by isolating binders toward Sortase A, a transamidase which is required for virulence of Staphylococcus aureus. The interaction of selected lantipeptides with Sortase A is highly dependent on the presence of a (2S,6R)-lanthionine in the peptide and an active conformation of the protein.

Advances in the directed evolution of proteins

“…A related approach created mRNA-displayed lantipeptides by using a translation system where lysine was substituted with 4-selenalysine, and inducing post-translational elimination via H 2 O 2 and dehydroalanine. This provided an alternative cyclization mechanism for a drug candidate library and yielded binders with low micromolar affinity for Sortase A [43•]. Similarly, a cDNA display library was cyclized [44] and a phage display library was bi-cyclized [45] through disulfide bonds formed in cysteine-rich peptides and used to select for peptide aptamers.…”

Encoded libraries of chemically modified peptides

“…They succeeded in isolating thrombin inhibitors based on peptides with natural (K d = 1.5 nM) and unnatural amino acids (K d = 20 nM). In another cyclization strategy Szostak and Seebeck incorporated 4-selenalysine into a peptide mRNA display library and transformed the residue by oxidative elimination to dehydroalanine so that it could react with a cysteine in the same peptide [37] (Figure 3g). In the Michael addition reaction, a new stereogenic center is generated resulting in two isomers.…”

Discovering functional, non-proteinogenic amino acid containing, peptides using genetic code reprogramming

“…This allows for the formation of lanthionine-like thioether (sulfide) bridged macrocyclic peptides. This was used in a selection against the protein Sortase A to produce a macrocyclic peptide with modest (3 µM K d ) binding affinity 28 .…”