Development of Uniform Ribosome Display Technology Enabling Easy and Efficient Identification of Full‐Length Proteins that Interact with Bioactive Small and Large Molecules

Abstract Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal‐length DNAs and mRNAs to improve molecular display principle for target identification. The equal‐length DNAs were designed to contain various coding sequences for full‐length proteins with molecular weights of up to 130 kDa and were used to synthesize equal‐length mRNAs, which allowed the formation of full‐length protein‐ribosome‐ equal‐length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein‐displaying ribosomal complexes were specifically selected through non‐covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full‐length proteins interacting with methotrexate or anti‐DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal‐length mRNAs encoding them were adjusted to 1/20,000 of the total equal‐length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.