In vitro display evolution of the PURE system-expressed TNFα-binding unnatural cyclic peptide containing an N-methyl-d-amino acid

Tumor necrosis factor-alpha (TNFα) is a multifunctional cytokine associated with inflammation, immune responses, and autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, psoriasis. In the present study, we performed in vitro selection, systematic evolution of ligands by exponential enrichment (SELEX) against human TNFα from mRNA-displayed peptide library prepared Escherichia coli-reconstituted cell-free transcription/translation system (PURE system) cyclized N-chloroacetyl-N-methyl-d-phenylalanine incorporated genetic code expansion (sense suppression). We identified novel TNFα-binding thioether-cyclized that contains an N-methyl-d-phenylalanine. Since cyclic structure presence N-methyl-d-amino acid can increase proteolytic stability, binding has potential to be used for therapeutic, research diagnostic applications.