title: An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study.
abstract: Intrinsically disordered proteins (IDPs) are an emerging concept. IDPs have high flexibility in their polypeptide chains, lacking a stable 3-dimensional structure. Because of the difficulty in performing X-ray crystallography for IDPs, nuclear magnetic resonance (NMR) spectroscopy is the first choice for atomic-level investigation of their nature. Given that isotopically labeled IDP samples are necessary for NMR study, a robust and cost-effective protocol for bacterial expression and purification of IDP is also needed. We employed the Npro(EDDIE)-autoprotease fusion protein system. Although IDPs are believed to be readily degraded by endogenous proteases when expressed inEscherichia coli, Npro-fused IDPs showed excellent resistance to degradation. Seven IDPs of uncharacterized function sampled from the human genome as well as 3 constructs from IDP regions derived from human FancM andThermococcus kodakarensisHef were prepared. We improved the protocol of refolding of Npro(EDDIE) to use dialysis, which is convenient for subsequent purification using reversed-phase (RP) HPLC. The method is robust and widely applicable to any IDP sample, promoting the acquisition of experimental data for IDPs in a high-throughput manner.
authors.affiliation: Laboratory of Structural Molecular Pharmacology; Graduate School of Pharmaceutical Sciences; Nagoya University; Furocho, Chikusa-ku, Nagoya, Japan; These authors equally contributed to the work.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Goda
authors.affiliation: Laboratory of Structural Molecular Pharmacology; Graduate School of Pharmaceutical Sciences; Nagoya University; Furocho, Chikusa-ku, Nagoya, Japan; These authors equally contributed to the work.
authors.forename: Naoki
authors.initials: N
authors.lastname: Matsuo
authors.affiliation: Laboratory of Structural Molecular Pharmacology; Graduate School of Pharmaceutical Sciences; Nagoya University; Furocho, Chikusa-ku, Nagoya, Japan; The Structural Biology Research Center and Division of Biological Science; Graduate School of Science; Nagoya University; Furocho, Nagoya, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tenno
authors.affiliation: Department of Bioscience and Biotechnology; Graduate School of Bioresource and Bioenvironmental Sciences and Faculty of Agriculture; Kyushu University ; Fukuoka, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Ishino
authors.affiliation: Department of Bioscience and Biotechnology; Graduate School of Bioresource and Bioenvironmental Sciences and Faculty of Agriculture; Kyushu University ; Fukuoka, Japan.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
authors.affiliation: Faculty of Engineering, Maebashi Institute of Technology ; Maebashi, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fukuchi
authors.affiliation: Department of Complex Systems Science; Graduate School of Information Sciences; Nagoya University ; Nagoya, Japan.
authors.forename: Motonori
authors.initials: M
authors.lastname: Ota
authors.affiliation: Laboratory of Structural Molecular Pharmacology; Graduate School of Pharmaceutical Sciences; Nagoya University; Furocho, Chikusa-ku, Nagoya, Japan; The Structural Biology Research Center and Division of Biological Science; Graduate School of Science; Nagoya University; Furocho, Nagoya, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Hiroaki