title: The silica-binding Si-tag functions as an affinity tag even under denaturing conditions.
abstract: We recently reported a one-step affinity purification method using a silica-binding protein, designated Si-tag, as a fusion partner and silica particles as the specific adsorbents (Ikeda et al., Protein Expr. Purif. 71 [2010] 91-95) [13]. In this study, we demonstrate that the Si-tag also binds to the silica surface even under denaturing conditions, thereby facilitating affinity purification of recombinant proteins from inclusion bodies. A fusion protein of the Si-tag and a biotin acceptor peptide (AviTag), which was expressed as inclusion bodies in Escherichia coli, was used as a model protein. To simplify our purification method, we disrupted recombinant E. coli cells by sonication in the presence of 8M urea with concomitant solubilization of the inclusion bodies. The fusion protein was recovered with a purity of 90 ± 3% and yield of 92 ± 6% from the cleared cell lysate. We also discuss the binding mechanism of the Si-tag to a silica surface in the presence of high concentrations of denaturant. We propose that the intrinsic disorder of the polycationic Si-tag polypeptide plays an important role in its binding to the silica surface under denaturing conditions.
authors.affiliation: Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan. ikedatakeshi@hiroshima-u.ac.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeda
authors.forename: Kei
authors.initials: K
authors.lastname: Motomura
authors.forename: Yuuya
authors.initials: Y
authors.lastname: Agou
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: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D010316:Particle Size; D011073:Polyamines / Q000378:metabolism; D000071228:Polyelectrolytes; D011485:Protein Binding; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012822:Silicon Dioxide / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013010:Sonication; D014508:Urea / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D011073:Polyamines; D000071228:Polyelectrolytes; D011993:Recombinant Fusion Proteins; D012822:Silicon Dioxide; D014508:Urea; C017071:biotin-binding proteins