GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE334nnn/GSE334901/primary200OKOthersynthetic constructOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE334901GEOGSEfalseHigh Throughput Characterization of Eukaryotic 2A-Like Peptides Identifies Novel Leucine-Associated Reduction in Protein AbundanceVirally-derived ribosomal skipping 2A peptides are a popular tool for protein co-production. Despite their use in over 9,000 publications, the biochemical and biophysical properties underlying the skipping mechanism remain largely unexplored. We identified 4,218 2A-like peptides originating from non-viral organisms. We developed and utilized the Trifluorescent Reporter fluorescent tool for high-throughput multiplexable analysis of ribosomal skipping, and tested 3,271 2A-like peptide sequences. We identified peptides that skipped, failed to skip, and skipped but failed to restart translation, in addition to peptides that induced a protein reduction phenotype. A poly-leucine stretch in a conserved alpha-helix N-terminal to the conserved GDxExNPGP motif drove both the skipping and reduction phenotypes, with the number and location of leucine residues being the determinant of which phenotype was observed. Analysis of the native eukaryotic protein contexts revealed that reduction may be harnessed as an expression regulator. The high-throughput approach used in this work greatly expands the functional knowledge of what biophysical and biochemical characteristics lead to ribosomal skipping, including a latent eukaryotic ‘leucine stall-helix’ motif.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 construct