[3S03e] 08. Functional Molecules and Materials for Biological Systems - Part 3

4:30 PM - 5:10 PM JST(7:30 AM - 8:10 AM UTC)

[3FMBS-10-KL] Ecobody and SMART: High-Throughput Cell-Free Systems for Rapid Protein Engineering

○Hideo Nakano¹ (1. Graduate School of Bioagricultural Sciences, Nagoya University (Japan))

Keywords:

cell-free protein synthesis,high-throughput screening,monoclonal antibody,D-amino acid oxidase,transglutaminase,single B cell

Cell-free protein synthesis systems are currently widely utilized not only in basic science but also in applied fields such as protein engineering, antibody engineering, and synthetic biology. We have established Ecobody technology, a novel high-throughput antibody generation system that uses reverse transcription, multiple rounds of PCR, and cell-free protein synthesis to produce significant quantities of antibody proteins from single B cells¹. This technology has been successfully commercialized by the startup company, iBody Inc. Within the Ecobody framework, a short translation-enhancing peptide (TEP) is critical for improving the productivity of antibody fragments. Recently, by using screening and bioinformatics, we have identified novel TEP sequences that further enhance the yield of difficult-to-express proteins². Furthermore, we developed an ultra-high-throughput in vitro selection platform for single enzyme molecules termed SMART (Single Molecule Assay on Ribonucleic acid by Translated product), designed for rapid and cost-effective enzyme evolution³. This system integrates mRNA display, next-generation sequencing, and bioinformatics, featuring an enzyme-specific selection module as a helper unit. This allows for the screening of various enzyme types under diverse experimental conditions. The platform is capable of handling exceptionally large libraries, with a diversity exceeding 10¹² unique variants. Until now we established the SMART platform for Schizosaccharomyces pombe D-amino acid oxidase (SpDAAO) and microbial transglutaminase (MTG) as model enzymes. For DAAO selection, engineered ascorbate peroxidase 2 was employed as a helper enzyme to detect hydrogen peroxide produced by DAAO activity, leading to the selective biotinylation of enzymatically active single-molecule display complexes. For MTG, a substrate peptide was designed to be accessible to the displayed MTG. These results highlight SMART as a fast, robust, and highly scalable platform for enzyme evolution, which can be readily customized for various enzyme chemistries by modifying the helper unit. These high-throughput selection and screening systems, particularly when integrated with bioinformatics, are expected to significantly contribute to the generation of novel functional proteins.
References
1. Ojima-Kato, T., Nagai, S., and Nakano, H. (2017) Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation. Sci. Rep. 7, 13979.
2. Ojima-Kato, T., Yokoyama, G., Nakano, H., Hamada, M., and Motono, C. (2026) Screening and machine learning-based prediction of translation-enhancing peptides that reduce ribosomal stalling in Escherichia coli. RSC Chem. Biol. 7, 58-66.
3. Munaweera, T. I. K., Odake N., Halim, H., Ikeda, K., Zhu, B., Camagna, M., Ito, T., Kitaguchi, T., Nemoto, N., Nakano, H., Damnjanović, J. Harnessing the Power of SMART Single-Molecule Display for Enzyme Evolution: A Focus on Oxidase. ACS Synth. Biol. 2026, XXXX, XXX, XXX-XXX https://doi.org/10.1021/acssynbio.5c00968