title: Quantitative evaluation of refolding conditions for a disulfide-bond-containing protein using a concise ¹⁸O-labeling technique.
abstract: A concise method was developed for quantifying native disulfide-bond formation in proteins using isotopically labeled internal standards, which were easily prepared with proteolytic ¹⁸O-labeling. As the method has much higher throughput to estimate the amounts of fragments possessing native disulfide arrangements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) than the conventional high performance liquid chromatography (HPLC) analyses, it allows many different experimental conditions to be assessed in a short time. The method was applied to refolding experiments of a recombinant neuregulin 1-β1 EGF-like motif (NRG1-β1), and the optimum conditions for preparing native NRG1-β1 were obtained by quantitative comparisons. Protein disulfide isomerase (PDI) was most effective at the reduced/oxidized glutathione ratio of 2:1 for refolding the denatured sample NRG1-β1 with the native disulfide bonds.
authors.affiliation: Laboratory of Proteomic Sciences, 21st Century COE Program, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Uchimura
authors.forename: Yusam
authors.initials: Y
authors.lastname: Kim
authors.forename: Takaaki
authors.initials: T
authors.lastname: Mizuguchi
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D020890:Neuregulin-1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010103:Oxygen Isotopes / Q000737:chemistry; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D004220:Disulfides; D020890:Neuregulin-1; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; C117467:neuregulin beta