Yuki Mochizuki
Saitama University
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Yuki Mochizuki.
Analytical Biochemistry | 2013
Yuki Mochizuki; Fumiaki Kohno; Koichi Nishigaki; Naoto Nemoto
In this paper, we demonstrate a novel pull-down method that dramatically reduces the cost and preparation time of a bait protein by cell-free translation with a puromycin linker. With the C-terminus of the bait protein linked to biotin through a puromycin molecule after the translation reaction and subsequent mRNA degradation by RNase, the prey protein was easily pulled down by streptavidin-coated magnetic beads in a test tube. Three fluorescent prey protein types were tested and confirmed by gel electrophoresis to be pulled down easily and rapidly, depending on their affinity.
Journal of Biotechnology | 2015
Yuki Mochizuki; Takeru Suzuki; Kenzo Fujimoto; Naoto Nemoto
cDNA display is a powerful in vitro display technology used to explore functional peptides and proteins from a huge library by in vitro selection. In addition to expediting the in vitro selection cycle by using cDNA display, easy and rapid functional analysis of selected candidate clones is crucial for high-throughput screening of functional peptides and proteins. In this report, a versatile puromycin-linker employing an ultrafast photo-cross-linker, 3-cyanovinylcarbazole nucleoside, is introduced. Its utility for both in vitro selection using cDNA display and protein-protein interaction analysis using a surface plasmon resonance (SPR) system is described. Using this versatile puromycin-linker, we demonstrated the model in vitro selection of the FLAG epitope and a SPR-based assay to measure the dissociation constant between the B domain of protein A and immunoglobulin G. Improvement of the puromycin-linker as described herein should make the cDNA display method easier to utilize for design of protein or peptide based affinity reagents.
Biological Procedures Online | 2013
Yuki Mochizuki; Shigefumi Kumachi; Koichi Nishigaki; Naoto Nemoto
BackgroundThe library size is critical for selection in evolutionary molecular engineering (directed evolution). Although cDNA display has become a promising in vitro display technology by overcoming the instability of mRNA display, it is hindered by low yields. In this study, we improved the yield of cDNA display molecules by carefully examining each step of the preparation process.FindingsWe found that steric hindrance of ribosomes binding to the mRNA-protein fusion molecules was interfering with biotin-streptavidin binding. Additionally, reducing buffer exchange by performing RNase digestion in the His-tag-binding buffer to release the cDNA display molecules improved their His-tag purification.ConclusionOur optimized conditions have improved the yield of cDNA display molecules by more than 10 times over currently used methods, making cDNA display more practically available in evolutionary molecular engineering.
Methods of Molecular Biology | 2012
Yuki Mochizuki; Naoto Nemoto
Protein scaffolds containing some disulfide bonds (e.g., Knottin, Kunitz domain, etc.) are promising candidates for molecular recognition. cDNA display has been developed to screen functional disulfide-rich peptide aptamers from a vast library by promoting disulfide bond shuffling after the synthesis of peptides in a cell-free translation system. Here we present a detailed protocol for the selection of disulfide-rich peptide aptamers against interleukin 6 receptor (IL-6R) from a 35-amino acid peptide library containing 32 amino acids in the random region, which is linked to its genotype by cDNA display.
Biology | 2015
Yutaro Tanemura; Yuki Mochizuki; Shigefumi Kumachi; Naoto Nemoto
Constrained peptides are an attractive class as affinity reagents or drug leads owing to their excellent binding properties. Many kinds of these peptides, such as cyclic peptides containing disulfide bridges, are found in nature or designed artificially by directed evolution. However, confirming the binding properties of the disulfide-rich peptides can be generally difficult, because of oxidative folding problems in the preparation steps. Therefore, a method for evaluating the binding properties of such peptides rapidly and easily is required. Here, we report an easy and rapid method for preparing biotin-attached peptides containing disulfide bridges or a chemical cross-linker using a cell-free translation system and a puromycin-linker, which is applicable to pull-down assays for protein (or peptide) molecular interaction analysis.
ACS Combinatorial Science | 2011
Yuki Mochizuki; Manish Biyani; Sachika Tsuji-Ueno; Miho Suzuki; Koichi Nishigaki; Yuzuru Husimi; Naoto Nemoto
Chemical Communications | 2014
Yuki Mochizuki; Koichi Nishigaki; Naoto Nemoto
Archive | 2010
Yuki Mochizuki; Naoto Nemoto; Koichi Nishigaki; 佑樹 望月; 直人 根本; 功一 西垣
Biochemical and Biophysical Research Communications | 2018
Takeru Suzuki; Yuki Mochizuki; Shinnosuke Kimura; Yoko Akazawa-Ogawa; Yoshihisa Hagihara; Naoto Nemoto
生物物理 | 2014
Yuki Mochizuki; Koichi Nishigaki; Naoto Nemoto