Nano‐sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing
Nano‐sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing
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DOI：
10.1002/bit.22240
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发表时间：
2009-05
影响因子：
3.8
通讯作者：
T. Yoshino;T. Nishimura;Tetsushi Mori;Shigeya Suzuki;H. Kambara;H. Takeyama;T. Matsunaga
中科院分区：
文献类型：
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作者：
T. Yoshino;T. Nishimura;Tetsushi Mori;Shigeya Suzuki;H. Kambara;H. Takeyama;T. Matsunaga
在分子生物学和应用生物科学中，对廉价和高通量DNA测序技术的需求很高。在这项研究中，开发了一种新型的纳米尺寸的磁性颗粒显示用于焦磷酸测序的酶，焦磷酸测序是一种相当新颖的生物发光DNA测序方法，该方法基于合成测序原理，采用级联的几种酶促反应。使用磁螺菌AMB-1的新型蛋白质展示系统，成功地将一种将PPi转化为ATP的高度耐热酶丙酮酸磷酸二激酶（PPDK）表达到细菌磁性颗粒（BacMP）上。评价了展示PPDK的BacMP（PPDK-BacMP）对pH和温度的酶稳定性，并显示了其广泛的性质。随后，将PPDK-BacMP应用于焦磷酸测序，并成功测序了靶寡核苷酸。显示在BacMP上展示的PPDK酶在每个序列反应中是可再循环的，因为它们可以通过磁力操纵。得出的结论是，纳米尺寸的PPDK-BacMP可用于缩小焦磷酸测序反应体积，从而允许高通量。酶的回收也被证明是有前途的，并适用于以低运行成本开发廉价的DNA测序。Biotechnol. Bioeng. 2009;103：130-137.© 2008 Wiley Periodicals，Inc.
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. Biotechnol. Bioeng. 2009;103: 130–137. © 2008 Wiley Periodicals, Inc.