Tumor necrosis factor-alpha (TNF alpha) is a multifunctional cytokine associated with inflammation, immune responses, and autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. In the present study, we performed in vitro selection, systematic evolution of ligands by exponential enrichment (SELEX) against human TNF alpha from mRNA-displayed peptide library prepared with Escherichia coli-reconstituted cell-free transcription/translation system (PURE system) and cyclized by N-chloroacetyl-N-methyl-D-phenylalanine incorporated by genetic code expansion (sense suppression). We identified a novel TNF alpha-binding thioether-cyclized peptide that contains an N-methyl-Dphenylalanine. Since cyclic structure and presence of an N-methyl-D-amino acid can increase proteolytic stability, the TNF alpha binding peptide has potential to be used for therapeutic, research and diagnostic applications. (C) 2020 Elsevier Inc. All rights reserved.