Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions
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- Takahashi Kazuki
- Graduate School of Science and Engineering, Saitama University
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- Sunohara Masato
- Graduate School of Science and Engineering, Saitama University
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- Terai Takuya
- Graduate School of Science and Engineering, Saitama University
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- Kumachi Shigefumi
- Graduate School of Science and Engineering, Saitama University
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- Nemoto Naoto
- Graduate School of Science and Engineering, Saitama University
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説明
<p>In vitro display technologies such as mRNA and cDNA display are powerful tools to create and select functional peptides. However, in some cases, efficiency of mRNA-protein fusion is very low, which results in decreased library size and lower chance of successful selection. In this study, to improve mRNA-protein fusion efficiency, we prepared an mRNA display library of a protein with random N- and C-terminal coding regions consisting of 12 nucleotides (i.e. four amino acids), and performed an electrophoresis mobility shift assay (EMSA)-based selection of successfully formed mRNA display molecules. A single-domain antibody (Nanobody, or VHH) was used as a model protein, and as a result, a pair of sequences was identified that increased mRNA-protein fusion efficiency of this protein by approximately 20%. Interestingly, enhancement of the fusion efficiency induced by the identified sequences was protein-specific, and different results were obtained for other proteins including VHHs with different CDRs. The results suggested that conformation of mRNA as a whole, rather than the amino acid sequence of the translated peptide, is an important factor to determine mRNA-protein fusion efficiency.</p>
収録刊行物
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- Biophysics and Physicobiology
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Biophysics and Physicobiology 14 (0), 23-28, 2017
一般社団法人 日本生物物理学会