title: Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.
abstract: We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification.
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan; Department of Botany and Microbiology, Faculty of Science, Minia University, Minia 61519, Egypt.
authors.forename: Mohamed A A
authors.initials: MA
authors.lastname: Abdelhamid
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Electronic address: ikedatakeshi@hiroshima-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeda
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Motomura
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Ishida
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Hirota
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
mesh_terms: D001120:Arginine / Q000378:metabolism; D001409:Bacillus cereus / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000302:isolation & purification* / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry* / Q000378:metabolism; D018707:Volcanic Eruptions
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012822:Silicon Dioxide; D001120:Arginine
keywords: Affinity purification; Arginine; Bacillus cereus CotB1; Fusion tag; Silica-binding peptide; Volcanic ash