Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing

Tomoko Yoshino, Taisei Nishimura, Tetsushi Mori, Shigeya Suzuki, Hideki Kambara, Haruko Takeyama, Tadashi Matsunaga

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)130-137
Number of pages8
JournalBiotechnology and Bioengineering
Volume103
Issue number1
DOIs
Publication statusPublished - 2009 May 1
Externally publishedYes

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Keywords

  • Bacterial magnetic particles
  • Pyrosequencing
  • Pyruvate phosphate dikinase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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