title: Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing.
abstract: There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yoshino
authors.forename: Taisei
authors.initials: T
authors.lastname: Nishimura
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Mori
authors.forename: Shigeya
authors.initials: S
authors.lastname: Suzuki
authors.forename: Hideki
authors.initials: H
authors.lastname: Kambara
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D042201:Magnetospirillum / Q000201:enzymology*; D053758:Nanoparticles; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D011772:Pyruvate, Orthophosphate Dikinase