title: Self-oriented immobilization of DNA polymerase tagged by titanium-binding peptide motif.
abstract: We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E. coli). The specific binding of the TBP-tagged DNA polymerase onto a TiO2 substrate was clearly monitored by surface plasmon resonance spectroscopy (SPR) and by surface potential detection with an extended-gate field effect transistor (FET). In the SPR analyses, constant quantities of the DNA polymerase were stably immobilized on the titanium substrate under flow conditions, regardless of the concentration of the DNA polymerase, and could be completely removed by a 4 M MgCl2 wash after measurement. The FET signal showed the contribution of the molecular charge in the TBP motif to the binding with TiO2. In addition, the TBP-tagged DNA polymerase-tethered TiO2 gate electrode enabled the effective detection of the positive charges of hydrogen ions produced by the DNA extension reaction, according to the FET principle. Therefore, the self-oriented immobilization platform based on the motif-inserted enzyme is suitable for the quick and stable immobilization of functional enzymes on biosensing devices.
authors.affiliation: Central Research Laboratory, Hitachi Ltd. 1-280 Higashi-Koigakubo, Kokubunji, Tokyo 185-8601, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Nishida
authors.forename: Taira
authors.initials: T
authors.lastname: Kajisa
authors.forename: Yuuya
authors.initials: Y
authors.lastname: Miyazawa
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tabuse
authors.forename: Takuya
authors.initials: T
authors.lastname: Yoda
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
authors.forename: Hideki
authors.initials: H
authors.lastname: Kambara
authors.forename: Toshiya
authors.initials: T
authors.lastname: Sakata
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D014025:Titanium / Q000737:chemistry*
chemical_list: D010455:Peptides; C009495:titanium dioxide; D014025:Titanium; D004259:DNA-Directed DNA Polymerase