A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis
A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis
复制标题
驱动三磷酸核苷再生和重构无细胞蛋白质合成的双功能多磷酸激酶
DOI：
10.1021/acssynbio.9b00456
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发表时间：
2019
影响因子：
4.7
通讯作者：
McGlynn, Shawn E.
中科院分区：
文献类型：
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作者：
Wang, Po-Hsiang;Fujishima, Kosuke;Berhanu, Samuel;Kuruma, Yutetsu;Jia, Tony Z.;Khusnutdinova, Anna N.;Yakunin, Alexander F.;McGlynn, Shawn E.
重构的无细胞蛋白质合成系统（例如，PURE系统）允许表达毒性蛋白、异源寡聚蛋白亚基和具有高水平同质性的非规范氨基酸的蛋白。在这些系统中，需要人工ATP/GTP再生系统来驱动蛋白质合成，这是使用三种激酶和磷酸肌酸完成的。在这里，我们证明了这三个激酶的替代与一个bifunctionalCytophaga hutchinsoniipolyphosphorylate激酶，磷酸化核苷在交换反应从多磷酸盐。优化的单激酶系统产生的最终sfGFP浓度（约530 μg/mL）超过三激酶系统（约400 μg/mL），在前90分钟内mRNA翻译速率快5倍。单激酶系统也与37 °C下热敏萤火虫荧光素酶的表达相容。潜在地，本文开发的单激酶核苷三磷酸再生方法可以扩展无细胞蛋白质合成系统的未来应用，并且可以用于驱动合成生物学中需要ATP和GTP的其他生化过程。
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 bifunctionalCytophaga hutchinsoniipolyphosphate 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.