[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-360] Screening of translation-enhancing peptides (TEPs) in Human cell-free translation system

○Yuma Nishikawa¹, Hideo Nakano¹, Teruyo Kato¹ (1. Grad. Sch. of Bioagr. Sci., Nagoya Univ. (Japan))

Keywords:

translation-enhancement,cDNA display,TEP

[Purpose] Efficient protein production is a fundamental requirement in biomanufacturing; however, translation efficiency often acts as a critical bottleneck, limiting overall yields. We have previously reported that short peptide tags consisting of only four amino acid residues, termed translation-enhancing peptides (TEPs), can enhance translation and increase protein production in Escherichia coli and yeast. Furthermore, we recently reported that these TEPs can alleviate ribosomal stalling induced by arrest peptides (APs) in E. coli. Despite these advances, TEPs that function effectively within mammalian expression systems have yet to be reported. This study aims to identify novel TEPs functional in mammalian cells to enhance productivity in mammalian biomanufacturing.[Method] We conducted screening using "cDNA display" with a human in vitro translation system to identify functional TEPs. A random library of 4 amino-acid peptides was constructed upstream of AP, with a selection tag placed downstream. In this design, the selection tag is translated only when the upstream 4 amino-acid peptide successfully alleviates the ribosomal stalling induced by the AP. By covalently linking the resulting peptide (phenotype) to their encoding cDNA (genotype) via a puromycin linker, we selectively enriched sequences capable of enhancing translation through the AP site. After multiple rounds of screening, the highly enriched TEP candidate sequences were identified by NGS analysis.[Results] Analysis of the NGS data revealed several TEP candidate sequences that were enriched through the cDNA display. The identification of these sequences marks a significant step toward developing novel platforms for optimizing protein production in mammalian biomanufacturing.