A pull-down method with a biotinylated bait protein prepared by cell-free translation using a puromycin linker

被引：9
|
作者：
Mochizuki, Yuki [1 ]
Kohno, Fumiaki [1 ]
Nishigaki, Koichi [1 ]
Nemoto, Naoto [1 ]
机构：
[1] Saitama Univ, Grad Sch Sci & Engn, Sakura Ku, Saitama 3388570, Japan
来源：
ANALYTICAL BIOCHEMISTRY | 2013年 / 434卷 / 01期
关键词：
Pull-down assay; Protein-protein interaction; In vitro selection; Puromycin; Cell-free translation; EPITOPE; ASSAYS;
D O I：
10.1016/j.ab.2012.10.041
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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. (C) 2012 Elsevier Inc. All rights reserved.
引用
收藏
页码：93 / 95
页数：3
相关论文
共 27 条
  • [1] Development of an Indole-Based Chemically Cleavable Linker Concept for Immobilizing Bait Compounds for Protein Pull-Down Experiments
    Sturm, Martin
    Leitner, Alexander
    Lindner, Wolfgang
    BIOCONJUGATE CHEMISTRY, 2011, 22 (02) : 211 - 217
  • [2] Single-Molecular Pull-Down for Quantifying Epigenetic Modifications in Cell-Free DNA
    Du, Yang
    Wang, Yongyao
    Liu, Jiyan
    Diao, Jiajie
    BIOPHYSICAL JOURNAL, 2019, 116 (03) : 139A - 139A
  • [3] Versatile C-Terminal Specific Biotinylation of Proteins Using Both a Puromycin-Linker and a Cell-Free Translation System for Studying High-Throughput Protein-Molecule Interactions
    Nemoto, Naoto
    Fukushima, Takayuki
    Kumachi, Shigefumi
    Suzuki, Miho
    Nishigaki, Koichi
    Kubo, Tai
    ANALYTICAL CHEMISTRY, 2014, 86 (17) : 8535 - 8540
  • [4] Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system
    Watanabe, Takayoshi
    Muranaka, Norihito
    Iijima, Issel
    Hohsaka, Takahlro
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2007, 361 (03) : 794 - 799
  • [5] Simple Screening Method for Autoantigen Proteins Using the N-Terminal Biotinylated Protein Library Produced by Wheat Cell-Free Synthesis
    Matsuoka, Kazuhiro
    Komori, Hiroaki
    Nose, Masato
    Endo, Yaeta
    Sawasaki, Tatsuya
    JOURNAL OF PROTEOME RESEARCH, 2010, 9 (08) : 4264 - 4273
  • [6] Fidelity rate determination in cell-free protein translation using LC-MS
    Heibeck, Tyler H.
    Yin, Gang
    Steiner, Alexander
    Roy, Mimi
    Murray, Christopher J.
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 2010, 239
  • [7] Improvement of translation efficiency in an Escherichia coli cell-free protein system using cysteine
    Shingaki, Tomoteru
    Nimura, Noriyuki
    PROTEIN EXPRESSION AND PURIFICATION, 2011, 77 (02) : 193 - 197
  • [8] Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system
    Chemla, Yonatan
    Ozer, Eden
    Schlesinger, Orr
    Noireaux, Vincent
    Alfonta, Lital
    BIOTECHNOLOGY AND BIOENGINEERING, 2015, 112 (08) : 1663 - 1672
  • [9] Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones
    Lyubov A. Ryabova
    Dominique Desplancq
    Alexander S. Spirin
    Andreas Plückthun
    Nature Biotechnology, 1997, 15 : 79 - 84
  • [10] Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones
    Ryabova, LA
    Desplancq, D
    Spirin, AS
    Pluckthun, A
    NATURE BIOTECHNOLOGY, 1997, 15 (01) : 79 - 84
123