title: Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions.
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.
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Sunohara
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Shigefumi
authors.initials: S
authors.lastname: Kumachi
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
keywords: Nanobody; electrophoresis mobility shift assay; in vitro translation; variable domain of heavy chain of heavy chain antibody