Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions

• Takahashi Kazuki

  Graduate School of Science and Engineering, Saitama University

• Sunohara Masato

  Graduate School of Science and Engineering, Saitama University

• Terai Takuya

  Graduate School of Science and Engineering, Saitama University

• Kumachi Shigefumi

  Graduate School of Science and Engineering, Saitama University

• Nemoto Naoto

  Graduate School of Science and Engineering, Saitama University

Abstract

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.

Journal

• Biophysics and Physicobiology

  Biophysics and Physicobiology 14 (0), 23-28, 2017

  The Biophysical Society of Japan

Keywords

• Nanobody
• variable domain of heavy chain of heavy chain antibody
• electrophoresis mobility shift assay
• in vitro translation

Details

• CRID: 1390001205764936960
• NII Article ID: 130005396150
• DOI: 10.2142/biophysico.14.0_23
• ISSN: 21894779
• Web Site: https://www.jstage.jst.go.jp/article/biophysico/14/0/14_23/_pdf
• Text Lang: en
• Data Source:
  • JaLC
  • Crossref
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed