title: Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.
abstract: We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.
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: Mohamed A A
authors.initials: MA
authors.lastname: Abdelhamid
authors.forename: Kei
authors.initials: K
authors.lastname: Motomura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeda
authors.forename: Takenori
authors.initials: T
authors.lastname: Ishida
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Hirota
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
mesh_terms: D000327:Adsorption; D001409:Bacillus cereus / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012822:Silicon Dioxide / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012822:Silicon Dioxide; D000097573:Red Fluorescent Protein