A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.,ACS Synthetic Biology

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A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2019-12-19 , DOI: 10.1021/acssynbio.9b00456
Po-Hsiang Wang ₁ , Kosuke Fujishima _{1,

2} , Samuel Berhanu ₁ , Yutetsu Kuruma _{1,

3} , Tony Z Jia _{1,

4} , Anna N Khusnutdinova ₅ , Alexander F Yakunin _{5,

6} , Shawn E McGlynn _{1,

4}

Affiliation

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 μg/mL) beyond that of the three-kinase system (∼400 μg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

中文翻译：

驱动核苷三磷酸再生和重构的无细胞蛋白合成的双功能多磷酸激酶。

重构的无细胞蛋白质合成系统（例如，PURE系统）可以表达有毒蛋白质，异源寡聚蛋白质亚基和具有非均质氨基酸且具有高同质性的蛋白质。在这些系统中，需要一个人工的ATP / GTP再生系统来驱动蛋白质合成，这需要使用三种激酶和磷酸肌酸来完成。在这里，我们展示了用一种双功能的Cytophaga hutchinsonii多磷酸激酶替代这三种激酶的过程，该激酶使多磷酸的交换反应中的核苷磷酸化。经过优化的单激酶系统产生的最终sfGFP浓度（〜530μg/ mL）超过了三激酶系统（〜400μg/ mL）的浓度，在前90分钟内mRNA的翻译速度提高了5倍。单激酶系统还与热敏感萤火虫萤光素荧光素酶在37°C下的表达兼容。潜在地，本文开发的单激酶三磷酸核苷再生方法可扩展无细胞蛋白质合成系统的未来应用，并可用于驱动需要ATP和GTP的合成生物学中的其他生化过程。

更新日期：2019-12-19

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