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.

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

“…Recently, the dNTP-induced fingers-closing transition was observed with a commercial biosensor that also revealed previously unidentified tight binding states for Taq and KF, and could presumably be expanded to measure analog incorporation [6]. The location of attachment is significant for the thermal stability of immobilized enzymes [53] and rapid, stable immobilization in a reproducible, specific orientation is required for consistent, sensitive detection [54]. …”

A molecular biology approach to protein coupling at a biosensor interface

“…This has been optimized for the purpose of irreversible immobilization to create FET devices [54]. Key residues in this peptide are considered to be the arginine in the first position and the aspartic acid at the fifth position which are proposed to point to the same face and interact with the -Oand -OH 2 + groups respectively, of the oxidized titanium.…”

Bio‐functionalized titanium surfaces with modified silk fibroin carrying titanium binding motif to enhance the ossific differentiation of MC3T3‐E1

“…Subsequent analyses have revealed that the N-terminal RKLPDA (termed minTBP-1) is both necessary and sufficient for the binding (Sano, Sasaki, & Shiba, 2005). This minTBP-1 motif has been widely employed as an appendix to provide an affinity to Ti in ferritin (Sano, Ajima, et al, 2005), artificial matrix proteins (Kokubun et al, 2008), BMP-2 (Kashiwagi et al, 2009), antimicrobial peptide (Geng et al, 2018;Yoshinari et al, 2010), calcification protein (Tsuji et al, 2010), DNA-polymerase (Nishida et al, 2015), carbon nanomaterial-binding peptide (Kokubun et al, 2018), and self-assembled chemical compounds (Ikemi et al, 2010). Interestingly, it has been revealed that the interaction between minTBP-1 and the surfaces of Ti is governed by reversible interactions (Brandt & Lyubartsev, 2015;Hayashi et al, 2009;Sano, Ajima, et al, 2005;Schneider & Ciacchi, 2012;Skelton et al, 2009).…”