Chemla Yonatan, Ozer Eden, Schlesinger Orr, Noireaux Vincent, Alfonta Lital
Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, 55401.
Biotechnol Bioeng. 2015 Aug;112(8):1663-72. doi: 10.1002/bit.25587. Epub 2015 Jun 16.
Cell-free protein synthesis offers a facile and rapid method for synthesizing, monitoring, analyzing, and purifying proteins from a DNA template. At the same time, genetic code expansion methods are gaining attention due to their ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins via ribosomal translation. These systems are based on the exogenous addition of an orthogonal translation system (OTS), comprising an orthogonal tRNA, and orthogonal aminoacyl tRNA synthetase (aaRS), to the cell-free reaction mixture. However, these components are unstable and their preparation is labor-intensive, hence introducing a major challenge to the system. Here, we report on an approach that significantly reduces the complexity, effort and time needed to express UAA-containing proteins while increasing stability and realizing maximal suppression efficiency. We demonstrate an endogenously introduced orthogonal pair that enables the use of the valuable yet insoluble pyrrolysyl-tRNA synthetase in a cell-free system, thereby expanding the genetic repertoire that can be utilized in vitro and enabling new possibilities for bioengineering. With the high stability and efficiency of our system, we offer an improved and accessible platform for UAA incorporation into proteins.
无细胞蛋白质合成提供了一种从DNA模板合成、监测、分析和纯化蛋白质的简便快速方法。同时，遗传密码扩展方法因其能够通过核糖体翻译将非天然氨基酸（UAA）位点特异性地掺入蛋白质中而受到关注。这些系统基于向无细胞反应混合物中外源添加一个正交翻译系统（OTS），该系统包括一个正交tRNA和一个正交氨酰tRNA合成酶（aaRS）。然而，这些组分不稳定且其制备过程 labor-intensive，因此给该系统带来了重大挑战。在此，我们报告了一种方法，该方法显著降低了表达含UAA蛋白质所需的复杂性、工作量和时间，同时提高了稳定性并实现了最大抑制效率。我们展示了一种内源性引入的正交对，其能够在无细胞系统中使用有价值但不溶性的吡咯赖氨酸tRNA合成酶，从而扩展了可在体外利用的遗传库，并为生物工程带来了新的可能性。凭借我们系统的高稳定性和效率，我们提供了一个用于将UAA掺入蛋白质的改进且易于使用的平台。
