Genetically Encoded Libraries of Nonstandard Peptides
Figure 2
Selection of nonstandard peptides from libraries generated by genetic code expansion. (a) Selection of backbone-cyclized L-p-benzoylphenylalanine-containing peptide inhibitors. Orthogonal tRNA(CUA)/aaRS pairs were expressed in E. coli in order to assign L-p-benzoylphenylalanine to the amber codon. SICROPPS was used to cyclize the peptides. (b) Proof-of-concept study of mRNA display with a nonproteinogenic amino acid. Biocytin was assigned to the amber codon by adding biocytin-tRNA(CUA) to a rabbit reticulocyte lysate. The peptides were expressed in mRNA display format and pulled down using streptavidin beads. (c) Selection of streptavidin binding non-standard peptides. Four nonproteinogenic amino acids were assigned to the amber codon and four-base codons (UAG, L-2-naphthylalanine; AGGU, L-p-biphenylalanine; CGGU, L-p-benzoylphenylalanine; GGGU, L-p-phenylazophenylalanine). All corresponding nonproteinogenic aa-tRNAs were added to an E. coli cell-free translation system. The peptide libraries were expressed in mRNA display format and used for in vitro selection against streptavidin. X represents one of the proteinogenic or nonproteinogenic amino acids, and Z represents one of the nonproteinogenic amino acids.
